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Sample records for bioluminescence reporter gene

  1. Infection with adenovirus-mediated luciferase reporter gene in mesenchymal stem cells and bioluminescence imaging

    International Nuclear Information System (INIS)

    Objective: To construct adenovirus vector containing firefly luciferase reporter gene (Ad-Luc) and infect bone marrow mesenchymal stem cells (BMSC), then to take bioluminescence imaging in vitro and in vivo for identification. Methods: The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV). It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing. PShuttle-CMV-Luc and backbone vector (pAdeno) were homologous recombined. Then the recombinant plasmid was packaged in HEK293 cells and the virus titer was detected. The BMSC were infected by the recombinant adenovirus. The bioluminescence imaging in vitro was performed to determine the best multiplicity of infection (MOI), and the relationship between bioluminescence intensity and MOI was analyzed by curve fitting regression analysis. Viability was evaluated via Trypan blue staining. The transfected BMSC (1 × 106) were implanted into the muscles of forelimb of SD rats,and then tracked by bioluminescence imaging in vivo. Cell viability was compared using two-way repeated measures analysis of variance between groups. Results: Enzyme digestion and sequence analysis indicated that Ad-Luc was successfully constructed. The virus titer was 1 × 1010 plaque forming unit (PFU)/ml. The bioluminescence detection in vitro showed that Ad-Luc could infect BMSC high efficiently to express luciferase and the best MOI was 50. The bioluminescence intensity enhanced with increase of MOI (R2 =0.98). No statistically significant difference was found in cell viability between transfected and untransfected BMSC at 1, 3, 5, 7 d. The cell survival rates were (92.5±2.3)% vs (94.1±1.8)%, (91.4±0.9)% vs (92.7±2.0)%, (92.1±1.6)% vs (93.3± 2.4)%, (91.9 ± 1.5)% vs (93.0 ± 3.1)%, respectively (F=4.38, P>0.05). The bioluminescence imaging in vivo showed that BMSC survived 1, 3, 7 d after implantation. However, bioluminescence signal decreased gradually over time

  2. 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...

  3. 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 g

  4. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    Science.gov (United States)

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  5. 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.

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

    International Nuclear Information System (INIS)

    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

  7. Detection of bacteria with bioluminescent reporter bacteriophage.

    Science.gov (United States)

    Klumpp, Jochen; Loessner, Martin J

    2014-01-01

    Bacteriophages are viruses that exclusively infect bacteria. They are ideally suited for the development of highly specific diagnostic assay systems. Bioluminescent reporter bacteriophages are designed and constructed by integration of a luciferase gene in the virus genome. Relying on the host specificity of the phage, the system enables rapid, sensitive, and specific detection of bacterial pathogens. A bioluminescent reporter phage assay is superior to any other molecular detection method, because gene expression and light emission are dependent on an active metabolism of the bacterial cell, and only viable cells will yield a signal. In this chapter we introduce the concept of creating reporter phages, discuss their advantages and disadvantages, and illustrate the advances made in developing such systems for different Gram-negative and Gram-positive pathogens. The application of bioluminescent reporter phages for the detection of foodborne pathogens is emphasized.

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

    International Nuclear Information System (INIS)

    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 gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated. Benzene ring 14C(U)-labeled d-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively. Radiolabeled and unlabeled d-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p=0.0002). Biodistribution studies in living mice after tail-vein injection of 14C-d-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of 14C-d-luciferin in firefly luciferase-expressing tissues could be observed in vivo. The data obtained with 14C-d-luciferin provide insights into the dynamics of d-luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and

  9. Construction of a Bioluminescent Reporter Strain To Detect Polychlorinated Biphenyls

    OpenAIRE

    Layton, A C; Muccini, M.; Ghosh, M. M.; Sayler, G. S.

    1998-01-01

    A bioluminescent reporter strain, Ralstonia eutropha ENV307(pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/lit...

  10. Amplitude metrics for cellular circadian bioluminescence reporters.

    Science.gov (United States)

    St John, Peter C; Taylor, Stephanie R; Abel, John H; Doyle, Francis J

    2014-12-01

    Bioluminescence rhythms from cellular reporters have become the most common method used to quantify oscillations in circadian gene expression. These experimental systems can reveal phase and amplitude change resulting from circadian disturbances, and can be used in conjunction with mathematical models to lend further insight into the mechanistic basis of clock amplitude regulation. However, bioluminescence experiments track the mean output from thousands of noisy, uncoupled oscillators, obscuring the direct effect of a given stimulus on the genetic regulatory network. In many cases, it is unclear whether changes in amplitude are due to individual changes in gene expression level or to a change in coherence of the population. Although such systems can be modeled using explicit stochastic simulations, these models are computationally cumbersome and limit analytical insight into the mechanisms of amplitude change. We therefore develop theoretical and computational tools to approximate the mean expression level in large populations of noninteracting oscillators, and further define computationally efficient amplitude response calculations to describe phase-dependent amplitude change. At the single-cell level, a mechanistic nonlinear ordinary differential equation model is used to calculate the transient response of each cell to a perturbation, whereas population-level dynamics are captured by coupling this detailed model to a phase density function. Our analysis reveals that amplitude changes mediated at either the individual-cell or the population level can be distinguished in tissue-level bioluminescence data without the need for single-cell measurements. We demonstrate the effectiveness of the method by modeling experimental bioluminescence profiles of light-sensitive fibroblasts, reconciling the conclusions of two seemingly contradictory studies. This modeling framework allows a direct comparison between in vitro bioluminescence experiments and in silico ordinary

  11. Construction of a bioluminescent reporter strain to detect polychlorinated biphenyls

    Energy Technology Data Exchange (ETDEWEB)

    Layton, A.C.; Muccini, M.; Ghosh, M.M.; Sayler, G.S. [Univ. of Tennessee, Knoxville, TN (United States)

    1998-12-01

    A bioluminescent reporter strain, Ralstonia eutropha ENV307 (pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.

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

    International Nuclear Information System (INIS)

    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 14C-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 [131I]FIAU (7.4 MBq/animal) or [124I]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 in the

  13. Bioluminescence Reporter Gene Imaging Characterize Human Embryonic Stem Cell-Derived Teratoma Formation

    OpenAIRE

    Su, Weijun; Zhou, Manqian; Zheng, Yizhou; Fan, Yan; HAN, ZHONGCHAO; Kong, Deling; Wu, Joseph C.; Xiang, Rong; Li, Zongjin

    2011-01-01

    Human embryonic stem (hES) cells are capable of differentiation into virtually all cell types and hold tremendous potential as cell sources for regenerative therapies. However, teratoma formation can be the main obstacle for hES cells therapy. In order to understand the biology and physiology of hES cells teratoma formation, we investigated the angiogenic process within teratomas and characterized teratoma cells. In this study, hES cells transduced with double fusion reporter gene that consis...

  14. Fluorescent and Bioluminescent Reporter Myxoviruses

    Science.gov (United States)

    Rostad, Christina A.; Currier, Michael C.; Moore, Martin L.

    2016-01-01

    The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo. PMID:27527209

  15. Fluorescent and Bioluminescent Reporter Myxoviruses.

    Science.gov (United States)

    Rostad, Christina A; Currier, Michael C; Moore, Martin L

    2016-01-01

    The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo. PMID:27527209

  16. Bioluminescence.

    Science.gov (United States)

    Jones, M. Gail

    1993-01-01

    Describes bioluminescence and the chemistry of how it occurs. Presents information for conducting the following classroom activities: (1) firefly mimic; (2) modeling deep-sea fish; (3) sea fireflies; and (4) the chemistry of light. (PR)

  17. Construction of a bioluminescence reporter plasmid for Francisella tularensis

    OpenAIRE

    Bina, Xiaowen R.; Miller, Mark A.; James E Bina

    2010-01-01

    A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia rese...

  18. Bioluminescent bacteria: lux genes as environmental biosensors Bactérias bioluminescentes: os genes lux como biosensores ambientais

    OpenAIRE

    Vânia da Silva Nunes-Halldorson; Norma Letícia Duran

    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...

  19. Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

    Science.gov (United States)

    Ideguchi, Yamato; Oshikoshi, Yuta; Ryo, Masashi; Motoki, Shogo; Kuwano, Takashi; Tezuka, Takafumi; Aoki, Setsuyuki

    2016-01-01

    We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

  20. Detection of traces of tetracyclines from fish with a bioluminescent sensor strain incorporating bacterial luciferase reporter genes.

    Science.gov (United States)

    Pellinen, Teijo; Bylund, Göran; Virta, Marko; Niemi, Anneli; Karp, Matti

    2002-08-14

    Bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline family of antimicrobial agents was optimized to work with fish samples. The biosensing strain contains a plasmid incorporating the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive element from transposon Tn10 (Korpela et al. Anal. Chem. 1998, 70, 4457-4462). The extraction procedure of oxytetracycline from rainbow trout (Oncorhynchus mykiss) tissue was optimized. There was neither need for centrifugation of homogenized tissue nor use of organic solvents. The lowest levels of detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50 microg/kg, respectively, in a 2-h assay. The optimized assay protocol was tested with fish that were given a single oral dose of high and low concentrations of oxytetracycline. The assay was able to detect oxytetracycline residues below the European Union maximum residue limits, and the results correlated well with those obtained by conventional HPLC (R = 0.81). PMID:12166964

  1. Bioluminescent reporter bacterium for toxicity monitoring in biological wastewater treatment systems

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, C.J.; Lajoie, C.A.; Layton, A.C.; Sayler, G.S.

    1999-01-01

    Toxic shock due to certain chemical loads in biological wastewater treatment systems can result in death of microorganisms and loss of floc structure. To overcome the limitations of existing approaches to toxicity monitoring, genes encoding enzymes for light production were inserted to a bacterium (Shk 1) isolated from activated sludge. The Shk 1 bioreporter indicated a toxic response to concentrations of cadmium, 2,4-dinitrophenol, and hydroquinone by reductions in initial levels of bioluminescence on exposure to the toxicant. The decrease in bioluminescence was more severe with increasing toxicant concentration. Bioluminescence did not decrease in response to ethanol concentrations up to 1,000 mg/L or to pH conditions between 6.1 and 7.9. A continuous toxicity monitoring system using this bioreporter was developed for influent wastewater and tested with hydroquinone. The reporter exhibited a rapid and proportional decrease in bioluminescence in response to increasing hydroquinone concentrations.

  2. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  3. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body. PMID:24058151

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

    International Nuclear Information System (INIS)

    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-[124I] iodouracil (124I - 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 (124I-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, 124I-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, 124I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by 124I-FIAU PET imaging. 124I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. 124I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials

  5. 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

  6. Destabilized bioluminescent proteins

    Science.gov (United States)

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  7. 腺病毒介导荧光素酶报告基因感染间充质干细胞的研究%Infection with adenovirus-mediated luciferase reporter gene in mesenchymal stem cells and bioluminescence imaging

    Institute of Scientific and Technical Information of China (English)

    王一帆; 夏睿; 郭玉林; 郜发宝

    2013-01-01

    目的 构建携带萤火虫荧光素酶(Luc)报告基因的腺病毒载体(Ad-Luc),研究其感染大鼠骨髓间充质干细胞(BMSC)后的体内外生物发光成像.方法 从psiCHECK-2质粒中用PCR扩增Luc基因,克隆入腺病毒穿梭载体pShuttle-CMV后行Nhe Ⅰ/Xba Ⅰ双酶切和测序鉴定.重组腺病毒穿梭载体与骨架载体pAdeno同源重组并包装纯化后,测定其病毒滴度.用重组Ad-Luc感染BMSC,行体外生物发光成像确定最佳感染复数(MOI),并采用曲线拟合回归分析生物发光强度与MOI的关系.以锥虫蓝染色法评价细胞活力变化,计算细胞存活率.将转染后BMSC(1×106个)植入SD大鼠前肢肌肉内,行体内生物发光成像.细胞存活率组间比较采用两因素重复测量资料方差分析.结果 经酶切和测序鉴定证明,Ad-Luc构建成功,病毒滴度为1×1010空斑形成单位(PFU)/ml.体外生物发光检测结果显示最佳MOI值为50,Ad-Luc可高效感染BMSC,使其表达Luc,且拟合曲线示细胞生物发光强度随MOI增加而增强(R2 =0.98).转染组和未转染组细胞培养1、3、5、7d时,细胞存活率分别为(92.5±2.3)%与(94.1±1.8)%、(91.4±0.9)%与(92.7±2.0)%、(92.1±1.6)%与(93.3±2.4)%、(91.9±1.5)%与(93.0±3.1)%,2组间细胞活力的差异无统计学意义(F=4.38,P>0.05).体内生物发光成像结果示BMSC移植1、3、7d后仍有存活,但随时间延长,生物发光信号逐渐减弱.结论 Luc报告基因通过腺病毒载体成功转入BMSC,实现了光学报告基因成像对移植干细胞的示踪.%Objective To construct adenovirus vector containing firefly luciferase reporter gene (AdLuc) and infect bone marrow mesenchymal stem cells (BMSC),then to take bioluminescence imaging in vitro and in vivo for identification.Methods The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV).It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing

  8. Transformation Experiment Using Bioluminescence Genes of "Vibrio fischeri."

    Science.gov (United States)

    Slock, James

    1995-01-01

    Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology. This laboratory experiment utilizes two plasmids of "Vibrio fischeri" in a transformation experiment. (LZ)

  9. Mechanisms of bioluminescence, chemiluminescence and of their regulation. Progress report, one year period through March 1976

    Energy Technology Data Exchange (ETDEWEB)

    Seliger, H H

    1976-01-01

    Progress is reported on a 10-yr study of the production and role of excited states in biological systems and the mechanisms involved in bioluminescence and chemoluminescence. An hypothesis of the origin of bioluminescence is presented that is based on the mixed function oxygenase reaction. Techniques of absolute measurements of light intensities and spectral composition were applied in studies of bioluminescence of marine dinoflagellates and the chemiluminescence of carcinogenic polycyclic aromatic hydrocarbons as the result of enzymatic hydroxylation. (CH)

  10. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

    Science.gov (United States)

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-01-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

  11. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy.

    Science.gov (United States)

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-03-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner.

  12. 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.

  13. Cellular bioluminescence imaging.

    Science.gov (United States)

    Welsh, David K; Noguchi, Takako

    2012-08-01

    Bioluminescence imaging of live cells has recently been recognized as an important alternative to fluorescence imaging. Fluorescent probes are much brighter than bioluminescent probes (luciferase enzymes) and, therefore, provide much better spatial and temporal resolution and much better contrast for delineating cell structure. However, with bioluminescence imaging there is virtually no background or toxicity. As a result, bioluminescence can be superior to fluorescence for detecting and quantifying molecules and their interactions in living cells, particularly in long-term studies. Structurally diverse luciferases from beetle and marine species have been used for a wide variety of applications, including tracking cells in vivo, detecting protein-protein interactions, measuring levels of calcium and other signaling molecules, detecting protease activity, and reporting circadian clock gene expression. Such applications can be optimized by the use of brighter and variously colored luciferases, brighter microscope optics, and ultrasensitive, low-noise cameras. This article presents a review of how bioluminescence differs from fluorescence, its applications to cellular imaging, and available probes, optics, and detectors. It also gives practical suggestions for optimal bioluminescence imaging of single cells.

  14. Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.

    Science.gov (United States)

    Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-09-01

    By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.

  15. 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.

  16. In vivo bioluminescence tomography with a blocking-off finite-difference SP3 method and MRI∕CT coregistration

    OpenAIRE

    Klose, Alexander D.; Beattie, Bradley J.; Dehghani, Hamid; Vider, Lena; Le, Carl; Ponomarev, Vladimir; Blasberg, Ronald

    2009-01-01

    Purpose: Bioluminescence imaging is a research tool for studying gene expression levels in small animal models of human disease. Bioluminescence light, however, is strongly scattered in biological tissue and no direct image of the light-emitting reporter probe’s location can be obtained. Therefore, the authors have developed a linear image reconstruction method for bioluminescence tomography (BLT) that recovers the three-dimensional spatial bioluminescent source distribution in small animals.

  17. Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

    Science.gov (United States)

    Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

    2012-06-01

    Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

  18. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    Science.gov (United States)

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  19. Quantitative bioluminescence imaging of mouse tumor models.

    Science.gov (United States)

    Tseng, Jen-Chieh; Kung, Andrew L

    2015-01-05

    Bioluminescence imaging (BLI) has become an essential technique for preclinical evaluation of anticancer therapeutics and provides sensitive and quantitative measurements of tumor burden in experimental cancer models. For light generation, a vector encoding firefly luciferase is introduced into human cancer cells that are grown as tumor xenografts in immunocompromised hosts, and the enzyme substrate luciferin is injected into the host. Alternatively, the reporter gene can be expressed in genetically engineered mouse models to determine the onset and progression of disease. In addition to expression of an ectopic luciferase enzyme, bioluminescence requires oxygen and ATP, thus only viable luciferase-expressing cells or tissues are capable of producing bioluminescence signals. Here, we summarize a BLI protocol that takes advantage of advances in hardware, especially the cooled charge-coupled device camera, to enable detection of bioluminescence in living animals with high sensitivity and a large dynamic range.

  20. 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...

  1. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  2. 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

    as it provides low background compared to other reporter genes such as green fluorescence protein (GFP). In cell culture, the luciferase can be used as reporter gene to see the effect of gene silencing. In the living fish, the bioluminescence signal detected is influenced by the melanin pigment. Timing between...

  3. Bioluminescence Imaging

    OpenAIRE

    Sadikot, Ruxana T.; Blackwell, Timothy S.

    2005-01-01

    Bioluminescence refers to the process of visible light emission in living organisms. Bioluminescence imaging is a powerful methodology that has been developed over the last decade as a tool for molecular imaging of small laboratory animals, enabling the study of ongoing biological processes in vivo. This form of optical imaging is low cost and noninvasive and facilitates real-time analysis of disease processes at the molecular level in living organisms. In this article, we provide a brief int...

  4. Multicolor Bioluminescence Obtained Using Firefly Luciferin.

    Science.gov (United States)

    Kiyama, Masahiro; Saito, Ryohei; Iwano, Satoshi; Obata, Rika; Niwa, Haruki; Maki, Shojiro A

    2016-01-01

    Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

  5. Joint effects of heavy metal binary mixtures on seed germination, root and shoot growth, bacterial bioluminescence, and gene mutation

    Institute of Scientific and Technical Information of China (English)

    In Chul Kong

    2013-01-01

    This investigation was to assess the joint effects of metal binary mixtures on seed germination,root and shoot growth,bacterial bioluminescence,and gene mutation based on the one toxic unit (1 TU) approach.Different sensitivities and orders of toxicity of metal mixtures were observed among the bioassays.In general,mostly additive or antagonistic effects were observed,while almost no synergistic effects by the binary metal mixtures in all bioassays.Therefore,the combined effects of heavy metals in the different bioassays were difficult to generalize since they were dependent on both chemical type and the organism used in each bioassay.However,these results indicate that a battery of bioassays with mixture chemicals as opposed to just a single assay with single metal is a better strategy for the bioassessment of environmental pollutants.

  6. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

    Directory of Open Access Journals (Sweden)

    Adil Bakayan

    Full Text Available Bioluminescence recording of Ca(2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET to the green fluorescent protein (GFP. This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+ in various cell compartments. In addition, they would also serve to monitor Ca(2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA showed the highest BRET efficiency (largest energy transfer critical distance R(0 and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+ oscillations in single HeLa cells expressing tdTA. Ca(2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+ activity of HeLa cells injected subcutaneously into mice, and Ca(2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+ sensor reported to date and is, therefore, a promising probe to study Ca(2+ dynamics in whole organisms or tissues expressing the transgene.

  7. Bioluminescence imaging in live cells and animals.

    Science.gov (United States)

    Tung, Jack K; Berglund, Ken; Gutekunst, Claire-Anne; Hochgeschwender, Ute; Gross, Robert E

    2016-04-01

    The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment.

  8. [Photoreactivating Activity of Bioluminescence: Repair of UV-damaged DNA of Escherichia coli Occurs with Assistance of lux-Genes of Marine Bacteria].

    Science.gov (United States)

    Zavilgelsky, G B; Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K Ss

    2015-01-01

    The UV resistance of luminescent bacteria Escherichia coli AB1886 uvrA6 (pLeo1) containing the plasmid with luxCDABE genes of marine bacteria Photobacterium leiognathi is approximately two times higher than the UV resistance of non-luminous bacteria E. coli AB1886 uvrA6. Introduction of phr::kan(r) mutations (a defect in the functional activity of photolyase) into the genome of E. coli AB1886 uvrA6 (pLeo1) completely removes the high UV resistance of the cells. Therefore, photoreactivation that involves bacterial photolyase contributes mainly to the bioluminescence-induced DNA repair. It is shown that photoreactivating activity of bioluminescence of P. leiognathi is about 2.5 times lower compared with that one induced by a light source with λ > 385 nm. It is also shown that an increase in the bioluminescence intensity, induced by UV radiation in E. coli bacterial cells with a plasmid containing the luxCD ABE genes under RecA-LexA-regulated promoters, occurs only 25-30 min later after UV irradiation of cells and does not contribute to DNA repair. A quorum sensing regulatory system is not involved in the DNA repair by photolyase.

  9. 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.

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

    International Nuclear Information System (INIS)

    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

  11. Combining fluorescence and bioluminescence microscopy.

    Science.gov (United States)

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  12. Lighting up bioluminescence with coelenterazine: strategies and applications.

    Science.gov (United States)

    Jiang, Tianyu; Du, Lupei; Li, Minyong

    2016-04-01

    Bioluminescence-based techniques, such as bioluminescence imaging, BRET and dual-luciferase reporter assay systems, have been widely used to examine a myriad of biological processes. Coelenterazine (CTZ), a luciferin or light-producing compound found in bioluminescent organisms, has sparked great curiosity and interest in searching for analogues with improved photochemical properties. This review summarizes the current development of coelenterazine analogues, their bioluminescence properties, and the rational design of caged coelenterazine towards biotargets, as well as their applications in bioassays. It should be emphasized that the design of caged luciferins can provide valuable insight into detailed molecular processes in organisms and will be a trend in the development of bioluminescent molecules.

  13. 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

  14. 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.

  15. The Chemical Basis of Fungal Bioluminescence.

    Science.gov (United States)

    Purtov, Konstantin V; Petushkov, Valentin N; Baranov, Mikhail S; Mineev, Konstantin S; Rodionova, Natalja S; Kaskova, Zinaida M; Tsarkova, Aleksandra S; Petunin, Alexei I; Bondar, Vladimir S; Rodicheva, Emma K; Medvedeva, Svetlana E; Oba, Yuichi; Oba, Yumiko; Arseniev, Alexander S; Lukyanov, Sergey; Gitelson, Josef I; Yampolsky, Ilia V

    2015-07-01

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  16. On-line monitoring of aerobic bioremediation with bioluminescent reporter microbes. Final report, July 1991--December 1994

    Energy Technology Data Exchange (ETDEWEB)

    Sayler, G.S.

    1995-03-01

    A critical issue in the biological characterization of contaminated sites and in the evaluation of relative bioremediation treatment efficiencies is the development of appropriate monitoring methods for the assessment of pollutant bioavailability and microbial in situ activity potential. In nature, pollutants are found dispersed among the solid, liquid and gaseous phases of the complex environments rendering the analytical estimation of their bioavailability and degradation more difficult and irrelevant. Ex situ and extractive analytical techniques have only been misrepresentative of the natural conditions and often resulted in inaccurate estimates of pollutants mass transfer. In this project, the bioluminescent bioreporter bacterium P. Fluorescens HK44 was integrated to an optical device, capable of conducting emitted light, and used as an online biosensor of naphthalene and salicylate. The physiological requirements of the bacteria and the physical limitations of the biosensor were also determined.

  17. 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.

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

    Science.gov (United States)

    Read, Hannah M.; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G.; Patrick, Wayne M.

    2016-01-01

    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. PMID:27366640

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

    Science.gov (United States)

    Read, Hannah M; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G; Patrick, Wayne M; Wiles, Siouxsie

    2016-01-01

    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. PMID:27366640

  20. Circadian Control Sheds Light on Fungal Bioluminescence

    OpenAIRE

    Oliveira, Anderson G.; Cassius V. Stevani; Waldenmaier, Hans E.; Viviani, Vadim; Emerson, Jillian M.; Loros, Jennifer J.; Jay C Dunlap

    2015-01-01

    Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi – only 71 species, all within the ~9000 fungi of the temperate and tropical Agaricales Order - are reported from among ~100,000 described fungal species [6,7]. All require oxygen [8] a...

  1. Circadian control sheds light on fungal bioluminescence.

    Science.gov (United States)

    Oliveira, Anderson G; Stevani, Cassius V; Waldenmaier, Hans E; Viviani, Vadim; Emerson, Jillian M; Loros, Jennifer J; Dunlap, Jay C

    2015-03-30

    Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.

  2. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Science.gov (United States)

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

  3. In Vivo Bioluminescence Imaging of the Murine Pathogen Citrobacter rodentium

    OpenAIRE

    Wiles, Siouxsie; Pickard, Karen M.; Peng, Katian; MacDonald, Thomas T.; Frankel, Gad

    2006-01-01

    Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal...

  4. 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

    2016-01-01

    Abstract 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 bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  5. Bioluminescence imaging characteristics and application

    International Nuclear Information System (INIS)

    Bioluminescence imaging (BLI) by luciferase gene marked cells or DNA, in the presence of ATP and oxygen, catalytic oxidation reaction of fluorescein luminescence. So that it can directly monitor in vivo cell activity and gene behavior. In this paper, by comparing the BLI and MRI, PET, radiography of the similarities and differences, as well as about their cancer, stem cells and immune cells transportation, apoptosis and other aspects of the application, in order to better provide the basis for promoting the application of BLI. (authors)

  6. A multimodality reporter gene for monitoring transplanted stem cells

    International Nuclear Information System (INIS)

    Introduction: The aim of this study is to explore the feasibility of a triple-fused reporter gene, termed TGF [herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescent protein (eGFP) and firefly luciferase (Fluc)], to monitor stem cells using multimodality molecular imaging. Methods: A recombinant adenovirus vector carrying the triple-fused reporter gene (Ad5-TGF) was constructed. Bone marrow mesenchymal stem cells (BMSCs) were transfected with different virus titers of Ad5-TGF [multiplicities of infection (MOIs) were 0, 50, 100, 150, 200 and 250]. The mRNA and protein expressions of HSV1-tk, eGFP and Fluc in the transfected BMSCs were evaluated using polymerase chain reaction and Western blot. After the transfection of the BMSCs with different virus titers of Ad5-TGF (MOIs were 25, 50, 75, 100 and 125), their uptake rates of 131I-FIAU were measured. Whole-body fluorescence, bioluminescence and micro-positron emission tomography (PET) images were acquired 1 day after the transfected BMSCs were injected into the left forelimb of rats. Results: After the transfection with different titers of Ad5-TGF, the positive transfection rate reached a peak (70%) when the MOI was 100. HSV1-tk, eGFP and Fluc mRNA and protein were detected in the Ad5-TGF-transfected BMSCs, which implies their successful transfection and expression. The BMSCs uptake of 131I-FIAU increased with the adenovirus titer and incubation time and reached a plateau (approximately 5.3%) after 3 h. Strong signals were observed in the injected left forearms in the fluorescence, bioluminescence and micro-PET images. Conclusions: A triple-fused reporter gene, TGF, can be used as a multifunctional molecular probe for multimodality imaging.

  7. Bioluminescence patterns among North American Armillaria species.

    Science.gov (United States)

    Mihail, Jeanne D

    2015-06-01

    Bioluminescence is widely recognized among white-spored species of Basidiomycota. Most reports of fungal bioluminescence are based upon visual light perception. When instruments such as photomultipliers have been used to measure fungal luminescence, more taxa have been discovered to produce light, albeit at a range of magnitudes. The present studies were undertaken to determine the prevalence of bioluminescence among North American Armillaria species. Consistent, constitutive bioluminescence was detected for the first time for mycelia of Armillaria calvescens, Armillaria cepistipes, Armillaria gemina, Armillaria nabsnona, and Armillaria sinapina and confirmed for mycelia of Armillaria gallica, Armillaria mellea, Armillaria ostoyae, and Armillaria tabescens. Emission spectra of mycelia representing all species had maximum intensity in the range 515-525 nm confirming that emitted light was the result of bioluminescence rather than chemiluminescence. Time series analysis of 1000 consecutive luminescence measurements revealed a highly significant departure from random variation. Mycelial luminescence of eight species exhibited significant, stable shifts in magnitude in response to a series of mechanical disturbance treatments, providing one mechanism for generating observed luminescence variation.

  8. Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

    OpenAIRE

    Jung, Song-Yi; Willard, Scott T

    2014-01-01

    Background The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investi...

  9. Using bioluminescence imaging in glioma research.

    Science.gov (United States)

    Luwor, Rodney B; Stylli, Stanley S; Kaye, Andrew H

    2015-05-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumour and has the worst prognosis. Over the last decade, the use of bioluminescence imaging technology has rapidly become widespread to further understand the mechanisms that drive GBM development and progression. Pre-clinical evaluation and optimisation of therapeutic efficacy in GBM research has also utilised this simple non-invasive technology. Here we summarise recent advances made in glioma biology and therapeutic intervention using bioluminescence imaging. This review also describes the current knowledge regarding the use of luciferase-based reporters in examining the role of specific cancer signalling cascades that promote glioma progression.

  10. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo

    Directory of Open Access Journals (Sweden)

    Rasko John EJ

    2010-11-01

    Full Text Available Abstract Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS, we generated clonal cell populations from a human breast cancer (MCF-7 and a mouse melanoma (B16-F10 cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments.

  11. Shedding light on bioluminescence regulation in Vibrio fischeri

    OpenAIRE

    Miyashiro, Tim; Ruby, Edward G.

    2012-01-01

    The bioluminescence emitted by the marine bacterium Vibrio fischeri is a particularly striking result of individual microbial cells coordinating a group behavior. The genes responsible for light production are principally regulated by the LuxR-LuxI quorum-sensing system. In addition to LuxR-LuxI, numerous other genetic elements and environmental conditions control bioluminescence production. Efforts to mathematically model the LuxR-LuxI system are providing insight into the dynamics of this a...

  12. Bioluminescence Imaging of Chlamydia muridarum Ascending Infection in Mice

    OpenAIRE

    Jessica Campbell; Yumeng Huang; Yuanjun Liu; Robert Schenken; Bernard Arulanandam; Guangming Zhong

    2014-01-01

    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 intrav...

  13. Targeted gene therapy and in vivo bioluminescent imaging for monitoring postsurgical recurrence and metastasis in mouse models of liver cancer.

    Science.gov (United States)

    He, Q; Yao, C L; Li, L; Xin, Z; Jing, Z K; Li, L X

    2016-01-01

    We investigated the effects of combined targeted gene therapy on recurrence and metastasis after liver cancer resection in nude mice. Twenty BALB/C mice were randomly divided into control and treatment groups with 10 mice in each group and a male/female ratio of 1:1. Luciferase gene-labeled human primary hepatic carcinoma cell line MHCC97-H was then used to prepare a carcinoma model. An optical in vivo imaging technique (OIIT) was used 10 days later to detect the distribution of tumor cells, followed by partial liver resection and gene therapy. In the treatment group, 100 mL phosphate-buffered saline (PBS) containing 1 x 1012 rAAV/AFP/IL-24 gene viral vectors was injected into liver sections and peritumoral posterior peritoneal tissues; in the control group, the same amount of PBS containing 1 x 1012 empty viral vectors was injected at the same sites. OIIT was then used to detect the in vivo tumor metastasis 21 days later. Luciferase gene-labeled human primary hepatic carcinoma cell line MHCC97-H successfully infected 20 nude mice, and OIIT showed that the two groups exhibited metastasis after local tumor resection, but there were more tumor cells in the control group (P < 0.05). rAAV/AFP/IL-24 gene therapy can inhibit recurrence after liver cancer resection. PMID:27525931

  14. Bioluminescent bioreporter integrated circuit

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L. (Knoxville, TN); Sayler, Gary S. (Blaine, TN); Paulus, Michael J. (Knoxville, TN)

    2000-01-01

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for environmental pollutant detection, oil exploration, drug discovery, industrial process control, and hazardous chemical monitoring.

  15. A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

    Science.gov (United States)

    Mezzanotte, Laura; Blankevoort, Vicky; Löwik, Clemens W G M; Kaijzel, Eric L

    2014-09-01

    Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

  16. Experimental approaches towards interpreting dolphin-stimulated bioluminescence.

    Science.gov (United States)

    Rohr, J; Latz, M I; Fallon, S; Nauen, J C; Hendricks, E

    1998-05-01

    Flow-induced bioluminescence provides a unique opportunity for visualizing the flow field around a swimming dolphin. Unfortunately, previous descriptions of dolphin-stimulated bioluminescence have been largely anecdotal and often conflicting. Most references in the scientific literature report an absence of bioluminescence on the dolphin body, which has been invariably assumed to be indicative of laminar flow. However, hydrodynamicists have yet to find compelling evidence that the flow remains laminar over most of the body. The present study integrates laboratory, computational and field approaches to begin to assess the utility of using bioluminescence as a method for flow visualization by relating fundamental characteristics of the flow to the stimulation of naturally occurring luminescent plankton. Laboratory experiments using fully developed pipe flow revealed that the bioluminescent organisms identified in the field studies can be stimulated in both laminar and turbulent flow when shear stress values exceed approximately 0.1 N m-2. Computational studies of an idealized hydrodynamic representation of a dolphin (modeled as a 6:1 ellipsoid), gliding at a speed of 2 m s-1, predicted suprathreshold surface shear stress values everywhere on the model, regardless of whether the boundary layer flow was laminar or turbulent. Laboratory flow visualization of a sphere demonstrated that the intensity of bioluminescence decreased with increasing flow speed due to the thinning of the boundary layer, while flow separation caused a dramatic increase in intensity due to the significantly greater volume of stimulating flow in the wake. Intensified video recordings of dolphins gliding at speeds of approximately 2 m s-1 confirmed that brilliant displays of bioluminescence occurred on the body of the dolphin. The distribution and intensity of bioluminescence suggest that the flow remained attached over most of the body. A conspicuous lack of bioluminescence was often observed on

  17. 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

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

    Science.gov (United States)

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

    2015-12-01

    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 Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.

  19. Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Ya-Ju Hsieh

    2014-01-01

    Full Text Available Multimodality imaging using noncytotoxic triple fusion (TF reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl, monomeric red fluorescence protein (mrfp, and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39 were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET. The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

  20. Circadian regulation of bioluminescence in Gonyaulax involves translational control.

    OpenAIRE

    Morse, D.; Milos, P M; Roux, E.; Hastings, J. W.

    1989-01-01

    A 10-fold circadian variation in the amount of luciferin binding protein (LBP) in the marine dinoflagellate Gonyaulax polyedra is reported. This protein binds and stabilizes luciferin, the bioluminescence substrate. In early night phase, when bioluminescence is increasing and LBP levels are rising in the cell, pulse labeling experiments show that LBP is being rapidly synthesized in vivo. At other times, the rate of LBP synthesis is at least 50 times lower, while the rate of synthesis of most ...

  1. 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.

  2. Validation of constitutively expressed bioluminescent Pseudomonas aeruginosa as a rapid microbiological quantification tool.

    Science.gov (United States)

    Shah, N; Naseby, D C

    2015-06-15

    Whole cell biosensors have been extensively used for monitoring toxicity and contamination of various compounds and xenobiotics in environmental biology and microbial ecology; their application in the pharmaceutical and cosmetics industries has been limited. According to several pharmacopoeias, pharmaceutical products must be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. However there is a lack of a validated bioluminescence method. Prototype whole cell microbial biosensors have already been developed in Pseudomonas aeruginosa ATCC 9027. Validation of the bioluminescent strains was performed in accordance with the pharmacopoeia, Parenteral Drug Association and International Organisation of Standardisation. These strains demonstrated that the bioluminescent method was accurate, precise and equivalent, as compared with plate counting at a range of 10(3)-10(7) CFU/mL. Percentage recoveries using the bioluminescent method were between 70% and 130% for all bioluminescent strains and therefore the bioluminescent method was accurate according to the criteria set in PDA technical report 33. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. The lower limit of detection was 10(3) CFU/mL. Two-way ANOVA showed no significant difference between the traditional plate counting and the novel bioluminescent method for all bioluminescent strains. The bioluminescent constructs passed/exceeded pharmacopoeia-specified criteria for range, limit of detection, accuracy, precision and equivalence.

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

    Science.gov (United States)

    Campbell, Jessica; Huang, Yumeng; Liu, Yuanjun; Schenken, Robert; Arulanandam, Bernard; Zhong, Guangming

    2014-01-01

    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.

  4. 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.

  5. BIOLUMINESCENCE IMAGING: PROGRESS AND APPLICATIONS

    OpenAIRE

    Badr, Christian E.; Tannous, Bakhos A

    2011-01-01

    Application of bioluminescence imaging has grown tremendously in the past decade and has significantly contributed to the core conceptual advances in biomedical research. This technology provides valuable means for monitoring of different biological processes for immunology, oncology, virology and neuroscience. In this review, we will discuss current trends in bioluminescence and its application in different fields with emphasis on cancer research.

  6. 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.

  7. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  8. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Close, Dan [University of Tennessee, Knoxville (UTK); Sayler, Gary Steven [ORNL; Xu, Tingting [ORNL; Ripp, Steven Anthony [ORNL

    2014-01-01

    Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitoring of signal intensity from any number of externally stimulated reporter proteins. While both viable methods, here we describe a novel technique that allows for the automated, non-destructive tracking of cellular population dynamics in real-time. This method, which relies on the detection of a continuous bioluminescent signal produced through expression of the bacterial luciferase gene cassette, provides a low cost, low time-intensive means for generating additional data compared to alternative methods.

  9. FNR-mediated regulation of bioluminescence and anaerobic respiration in the light-organ symbiont Vibrio fischeri

    OpenAIRE

    Septer, Alecia N.; Bose, Jeffrey L.; Dunn, Anne K.; Stabb, Eric V.

    2010-01-01

    Vibrio fischeri induces both anaerobic respiration and bioluminescence during symbiotic infection. In many bacteria, the oxygen-sensitive regulator FNR activates anaerobic respiration, and a preliminary study using the light-generating lux genes from V. fischeri MJ1 cloned in Escherichia coli suggested that FNR stimulates bioluminescence. To test for FNR-mediated regulation of bioluminescence and anaerobic respiration in V. fischeri, we generated fnr mutants of V. fischeri strains MJ1 and ES1...

  10. Chemiluminescence and bioluminescence microbe detection

    Science.gov (United States)

    Taylor, R. E.; Chappelle, E.; Picciolo, G. L.; Jeffers, E. L.; Thomas, R. R.

    1978-01-01

    Automated biosensors for online use with NASA Water Monitoring System employs bioluminescence and chemiluminescence techniques to rapidly measure microbe contamination of water samples. System eliminates standard laboratory procedures requiring time duration of 24 hours or longer.

  11. Bioluminescence tomography with Gaussian prior

    OpenAIRE

    Gao, Hao; Zhao, Hongkai; Cong, Wenxiang; Wang, Ge

    2010-01-01

    Parameterizing the bioluminescent source globally in Gaussians provides several advantages over voxel representation in bioluminescence tomography. It is mathematically unique to recover Gaussians [Med. Phys. 31(8), 2289 (2004)] and practically sufficient to approximate various shapes by Gaussians in diffusive medium. The computational burden is significantly reduced since much fewer unknowns are required. Besides, there are physiological evidences that the source can be modeled by Gaussians....

  12. In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter

    OpenAIRE

    Van de Bittner, Genevieve C.; Dubikovskaya, Elena A.; Bertozzi, Carolyn R.; Chang, Christopher J.

    2010-01-01

    Living organisms produce hydrogen peroxide (H2O2) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H2O2 on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-...

  13. 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 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 incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  14. 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.

  15. Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

    OpenAIRE

    Yu Wang; Hidefumi Akiyama; Kanako Terakado; Toru Nakatsu

    2013-01-01

    Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and fo...

  16. Radionuclide reporter gene imaging for cardiac gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Inubushi, Masayuki [Hokkaido University Graduate School of Medicine, Department of Molecular Imaging, Sapporo (Japan); Tamaki, Nagara [Hokkaido University Graduate School of Medicine, Department of Nuclear Medicine, Sapporo (Japan)

    2007-06-15

    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.)

  17. Exploiting in vitro and in vivo bioluminescence for the implementation of the three Rs principle (replacement, reduction, and refinement) in drug discovery.

    Science.gov (United States)

    Michelini, Elisa; Cevenini, Luca; Calabretta, Maria Maddalena; Calabria, Donato; Roda, Aldo

    2014-09-01

    Bioluminescence-based analytical tools are suitable for high-throughput and high-content screening assays, finding widespread application in several fields related to the drug discovery process. Cell-based bioluminescence assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of drug screening. Reporter gene technology and the bioluminescence resonance energy transfer principle are widely used, and receptor binding studies of new agonists/antagonists for a variety of human receptors expressed in different cell lines can be performed. Moreover, bioluminescence can be used for in vitro and in vivo real-time monitoring of pathophysiological processes within living cells and small animals. New luciferases and substrates have recently arrived on the market, further expanding the spectrum of applications. A new generation of probes are also emerging that promise to revolutionize the preclinical imaging market. This formidable toolbox is demonstrated to facilitate the implementation of the three Rs principle in the early drug discovery process, in compliance with ethical and responsible research to reduce cost and improve the reliability and predictability of results.

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

    Science.gov (United States)

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

    2016-01-08

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

  19. Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

    OpenAIRE

    Kondo, T.; Ishiura, M

    1994-01-01

    Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent ...

  20. Regulated bioluminescence as a tool for bioremediation process monitoring and control of bacterial cultures

    Science.gov (United States)

    Burlage, Robert S.; Heitzer, Armin; Digrazia, Philip M.

    1991-01-01

    An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed.

  1. ATP binding cassette transporters modulate both coelenterazine- and D-luciferin- based bioluminescence imaging

    OpenAIRE

    Huang, Ruimin; Vider, Jelena; Serganova, Inna; Blasberg, Ronald G.

    2011-01-01

    Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI-readout-intensity from intact living cells.

  2. A novel reconstruction algorithm for bioluminescent tomography based on Bayesian compressive sensing

    Science.gov (United States)

    Wang, Yaqi; Feng, Jinchao; Jia, Kebin; Sun, Zhonghua; Wei, Huijun

    2016-03-01

    Bioluminescence tomography (BLT) is becoming a promising tool because it can resolve the biodistribution of bioluminescent reporters associated with cellular and subcellular function through several millimeters with to centimeters of tissues in vivo. However, BLT reconstruction is an ill-posed problem. By incorporating sparse a priori information about bioluminescent source, enhanced image quality is obtained for sparsity based reconstruction algorithm. Therefore, sparsity based BLT reconstruction algorithm has a great potential. Here, we proposed a novel reconstruction method based on Bayesian compressive sensing and investigated its feasibility and effectiveness with a heterogeneous phantom. The results demonstrate the potential and merits of the proposed algorithm.

  3. 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.

  4. Standardized application of yeast bioluminescent reporters as endocrine disruptor screen for comparative analysis of wastewater effluents from membrane bioreactor and traditional activated sludge.

    Science.gov (United States)

    Wang, Jun; Eldridge, Melanie; Menn, Fu-min; Dykes, Todd; Sayler, Gary

    2015-12-01

    A standardized protocol is demonstrated for bioluminescent strains Saccharomyces cerevisiae BLYES, BLYAS and BLYR as high-throughput screening tools to monitor the estrogenic, androgenic and toxic potencies in wastewater. The sensitivity and reproducibility of the assay in wastewater monitoring was evaluated for 7 day semi-continuous batch reactor using activated sludge with hormones spiked raw sewage. Yeast bioluminescent assay successfully captured the rapid removal of estrogenic and androgenic activities in the bioreactors, and demonstrated rapid response (≤4 h) with good reproducibility. This standardized protocol was then applied in a 12 months monitoring of the effluent of a WWTP located at Powell, TN, USA featuring parallel-operated full-scale membrane bioreactor (MBR) and traditional activated sludge (TAS) treatment. Monitoring results showed that estrogenic activity was persistent in all TAS and most MBR effluent samples, while residual androgenic activity was non-detectable throughout the monitored period. The estrogenic equivalents (EEQ) in TAS effluent ranged from 21.61 ng/L to 0.04 pg/L and averaged 3.25 ng/L. The EEQ in MBR effluent ranged from 2.88 ng/L to 0.0134 pg/L and averaged ~10 fold less (0.32 ng/L) than TAS. Despite the large temporal variation, MBR effluent EEQ was consistently lower than TAS on any given sampling date. Most MBR effluent samples also exhibited less cytotoxicity than TAS. Further analysis did not demonstrate significant correlation between effluent EEQ level and WWTP operational parameters including MLSS, SRT, HRT and BOD.

  5. Comprehensive assessment of host responses to ionizing radiation by nuclear factor-κB bioluminescence imaging-guided transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Chung-Ta Chang

    Full Text Available The aim of this study was to analyze the host responses to ionizing radiation by nuclear factor-κB (NF-κB bioluminescence imaging-guided transcriptomic tool. Transgenic mice carrying the NF-κB-driven luciferase gene were exposed to a single dose of 8.5 Gy total-body irradiation. In vivo imaging showed that a maximal NF-κB-dependent bioluminescent intensity was observed at 3 h after irradiation and ex vivo imaging showed that liver, intestine, and brain displayed strong NF-κB activations. Microarray analysis of these organs showed that irradiation altered gene expression signatures in an organ-specific manner and several pathways associated with metabolism and immune system were significantly altered. Additionally, the upregulation of fatty acid binding protein 4, serum amyloid A2, and serum amyloid A3 genes, which participate in both inflammation and lipid metabolism, suggested that irradiation might affect the cross pathways of metabolism and inflammation. Moreover, the alteration of chemokine (CC-motif ligand 5, chemokine (CC-motif ligand 20, and Jagged 1 genes, which are involved in the inflammation and enterocyte proliferation, suggested that these genes might be involved in the radiation enteropathy. In conclusion, this report describes the comprehensive evaluation of host responses to ionizing radiation. Our findings provide the fundamental information about the in vivo NF-κB activity and transcriptomic pattern after irradiation. Moreover, novel targets involved in radiation injury are also suggested.

  6. 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.

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

    International Nuclear Information System (INIS)

    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.

  8. A Nisin Bioassay Based on Bioluminescence

    OpenAIRE

    Wahlström, G.; Saris, P. E. J.

    1999-01-01

    A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.

  9. Protein-protein complexation in bioluminescence

    OpenAIRE

    Titushin, Maxim S.; Feng, Yingang; Lee, John; Vysotski, Eugene S.; Liu, Zhi-jie

    2011-01-01

    In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an “accessory protein” whereby a stored substrate is efficiently delivered to the bioluminescent enzyme lucife...

  10. Hydromechanical stimulation of bioluminescent plankton.

    Science.gov (United States)

    Blaser, Stefan; Kurisu, Futoshi; Satoh, Hiroyasu; Mino, Takashi

    2002-01-01

    The response of the bioluminescent dinoflagellate Pyrocystis fusiformis was investigated for different hydraulic conditions ('hydromechanical stimulation'). Pipe flow and oscillating shear produced luminescence, whereas changes in hydrostatic pressure were not stimulating. More intense fluid motion led to higher intensity, mainly due to a higher probability of cell response. The organism was also able to emit light in a glucose-salt mixture. The experiments suggest that the cells are effectively stimulated if the flow conditions change in time.

  11. Luciferase bioluminescence imaging monitoring gene therapeutic effect of apoptosis-inducing ligand for lung cancer A549 cells nude mice transplantation tumor in vivo

    International Nuclear Information System (INIS)

    Objective: To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand (hTRAIL) in vivo,by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase (luc) gene (ad-luc-hTRAIL), in which luc was used as reporter gene. Methods: Lung cancer A549 cell xenografts in 16 nude mice models were established in subcutaneous inoculation way, the adenovirus vectors (ad-luc-hTRAIL, ad-hTRAIL, ad-luc) and phosphate buffer saline (PBS) (n=4) as control were injected into tumor respectively. The size of the tumor was measured at different time points (4, 7, 10, 14, 21, 28 d) after injection. The activity of luciferase in surface of the tumor was detected in vivo by using high-sensitivity cooled-charged coupled device (CCD) camera. The expression of hTRAIL was demonstrated by immunohistochemistry staining after sacrificing the animals at different time points, and immunohistochemical scores (IHS) were measured. The apoptosis rate of tumor cells was detected by using TUNEL and calculated. Analysis of variance, the paired t test and linear correlation analysis was used for the statistics. Results: The growing speed of tumour xenografts was more slowly in ad-luc-hTRAIL and ad-hTRAIL groups than PBS group (t=2.71, 2.72, P<0.05). The tumor volumes of ad-luc-hTRAIL, ad-hTRAIL, ad-luc and PBS groups 28 days after injection were (208.4 ± 42.3), (181.5 ±23.9), (403.1 ± 54.0) and (427.0 ± 59.3) mm3, respectively. There was no significant difference between ad-luc group and PBS group (t=2.07, P>0.05). The expression of luciferase in ad-luc-hTRAIL group reached its peak at 7th day (1.37 ± 1.04), and then decreased quickly. The IHS and apoptosis rate in ad-luc-hTRAIL and ad-hTRAIL groups reached their peaks at 7th day, the peak values of IHS were 6.25 ±2.06 and 6.5 ± 2.89, the peak values of apoptosis rate were (60.75 ± 8.06)% and (61.50 ± 8.47)%,respectively. The amount of luciferase expression (absolute number of

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

    Science.gov (United States)

    Mahdi, Min Y.; Gonzalez-Gomez, Ignacio; Asgharzadeh, Shahab; D’Apuzzo, Massimo; Erdreich-Epstein, Anat; Moats, Rex A.

    2016-01-01

    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 subsequent

  13. An improved single-step lysis protocol to measure luciferase bioluminescence in Plasmodium falciparum

    OpenAIRE

    Hasenkamp Sandra; Wong Eleanor H; Horrocks Paul

    2012-01-01

    Abstract This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore abs...

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

    OpenAIRE

    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 fl...

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

    Energy Technology Data Exchange (ETDEWEB)

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung [School of Midicine, Chonnam National University, Gwangju (Korea, Republic of)

    2004-07-01

    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{sup 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.

  16. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    Science.gov (United States)

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  17. Bioluminescence imaging of cord blood derived mesenchymal stem cell transplanatation into myocardium

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Ahn, Young Keun; Moon, Sung Min; Lim, Sang Yup; Yun, Kyung Ho; Heo, Young Jun; Song, Ho Chun; Jeong, Myung Ho; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of)

    2004-07-01

    The conventional method of analyzing myocardial cell transplanation relies on postmortem histology. We sought to demonstrate the feasibility of longitudinal monitoring transplanted cell survival in living animals using optical imaging techniques. Umblical cord blood was collected upon delivery with informed consent. Umblical mononuclear cells were obtained by negative immuno-depletion of CD3, CD14, CD19, CD38, CD66b, and glycophorin- A positive cells, followed by Ficoll- Paque density gradient centrifugation, and plated in non-coated tissue culture flasks in expansion medium. Cells were allowed to adhere overnight, thereafter non-adherent cells were washed out with medium changes. After getting the MSCs, they were transfected [multiplicity of infection (MOl) = 40) with Ad-CMV-Fluc overnight. Rats (n=4) underwent intramyocardial injection of 5 x 10{sup 5} MSCs expressing firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was performed using the charged-coupled device camera (Xenogen) from the 1st day of transplantion. Cardiac bioluminescence signals were present from 2nd day of transplantation. Cardiac signals were clearly present at day 2 (9.2x10{sup 3}p/s/cm{sup 2}/sr). The signal reduced from day 3. The locations, magnitude, and survival duration of cord blood derived MSCs were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation.

  18. Bioluminescence imaging of cord blood derived mesenchymal stem cell transplanatation into myocardium

    International Nuclear Information System (INIS)

    The conventional method of analyzing myocardial cell transplanation relies on postmortem histology. We sought to demonstrate the feasibility of longitudinal monitoring transplanted cell survival in living animals using optical imaging techniques. Umblical cord blood was collected upon delivery with informed consent. Umblical mononuclear cells were obtained by negative immuno-depletion of CD3, CD14, CD19, CD38, CD66b, and glycophorin- A positive cells, followed by Ficoll- Paque density gradient centrifugation, and plated in non-coated tissue culture flasks in expansion medium. Cells were allowed to adhere overnight, thereafter non-adherent cells were washed out with medium changes. After getting the MSCs, they were transfected [multiplicity of infection (MOl) = 40) with Ad-CMV-Fluc overnight. Rats (n=4) underwent intramyocardial injection of 5 x 105 MSCs expressing firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was performed using the charged-coupled device camera (Xenogen) from the 1st day of transplantion. Cardiac bioluminescence signals were present from 2nd day of transplantation. Cardiac signals were clearly present at day 2 (9.2x103p/s/cm2/sr). The signal reduced from day 3. The locations, magnitude, and survival duration of cord blood derived MSCs were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation

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

    International Nuclear Information System (INIS)

    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 mm3 (R 2=0.907). γ Scintigraphy combined with [131I]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

  20. A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging.

    Science.gov (United States)

    Jathoul, Amit P; Grounds, Helen; Anderson, James C; Pule, Martin A

    2014-11-24

    Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λ(max)=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

  1. REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    Science.gov (United States)

    This review of the recent literature on environmental applications of bioluminescence systems will focus on in vivo and in vitro bioluminescence methods that have been utilized to elucidate properties of chemicals, toxic and mutagenic effects, and to estimate biomass. he unifying...

  2. 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 Photoacoustic imaging could resolve the two tubes of 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

  3. Understanding bioluminescence in dinoflagellates — how far have we come?

    OpenAIRE

    Martha Valiadi; Debora Iglesias-Rodriguez

    2013-01-01

    Some dinoflagellates possess the remarkable genetic, biochemical, and cellular machinery to produce bioluminescence. Bioluminescent species appear to be ubiquitous in surface waters globally and include numerous cosmopolitan and harmful taxa. Nevertheless, bioluminescence remains an enigmatic topic in biology, particularly with regard to the organisms’ lifestyle. In this paper, we review the literature on the cellular mechanisms, molecular evolution, diversity, and ecology of bioluminescence ...

  4. Bioluminescence tomography based on the phase approximation model

    OpenAIRE

    Cong, W; Wang, G.

    2010-01-01

    A reconstruction method of bioluminescence sources is proposed based on a phase approximation model. Compared with the diffuse approximation, this phase approximation model more correctly predicts bioluminescence photon propagation in biological tissues, so that bioluminescence tomography can accurately locate and quantify the distribution of bioluminescence sources. The compressive sensing (CS) technique is applied to regularize the inverse source reconstruction to enhance numerical stabilit...

  5. Detection of a bioluminescent milky sea from space

    OpenAIRE

    Miller, Steven D.; Haddock, Steven H. D.; Elvidge, Christopher D.; Thomas F. Lee

    2005-01-01

    On many occasions over the centuries, mariners have reported witnessing surreal nocturnal displays where the surface of the sea produces an intense, uniform, and sustained glow that extends to the horizon in all directions. Although such emissions cannot be fully reconciled with the known features of any light-emitting organism, these so-called “milky seas” are hypothesized to be manifestations of unusually strong bioluminescence produced by colonies of bacteria in association with a microalg...

  6. Mathematical Study and Numerical Simulation of Multispectral Bioluminescence Tomography

    OpenAIRE

    Weimin Han; Wenxiang Cong; Ge Wang

    2006-01-01

    Multispectral bioluminescence tomography (BLT) attracts increasingly more attention in the area of optical molecular imaging. In this paper, we analyze the properties of the solutions to the regularized and discretized multispectral BLT problems. First, we show the solution existence, uniqueness, and its continuous dependence on the data. Then, we introduce stable numerical schemes and derive error estimates for numerical solutions. We report some numerical results to illust...

  7. Bioluminescence in vivo imaging of autoimmune encephalomyelitis predicts disease

    OpenAIRE

    Steinman Lawrence; Ho Peggy; Luo Jian; Wyss-Coray Tony

    2008-01-01

    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 co...

  8. Synthetic Bioluminescent Coelenterazine Derivatives.

    Science.gov (United States)

    Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

    2016-01-01

    The development of coelenterazine (CTZ) derivatives resulting in superior optical characteristics is an efficient method to extend the range of its possible applications. Here, we describe the synthesis of three C-6 substituted CTZ derivatives retaining the recognition by Renilla luciferase (RLuc) and its derivatives. The novel derivatives are useful as bright blue-shifted CTZ derivatives, which can be used as an alternative to hitherto reported compound DeepBlueC™. PMID:27424892

  9. Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

    Science.gov (United States)

    Chan, Harvey Y S; Cheung, Man Chun; Gao, Yi; Miller, Andrew L; Webb, Sarah E

    2016-08-01

    In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation. PMID:27430888

  10. QM/MM study on the light emitters of aequorin chemiluminescence, bioluminescence, and fluorescence: a general understanding of the bioluminescence of several marine organisms.

    Science.gov (United States)

    Chen, Shu-Feng; Ferré, Nicolas; Liu, Ya-Jun

    2013-06-24

    Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases).

  11. Bioluminescence imaging to monitor the prolongation of stem cell survival by pharmaceutical intervention

    Energy Technology Data Exchange (ETDEWEB)

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Ahn, Young Keun; Kim, Yong Sook; Joo, Soo Yeon; Hong, Moon Hwa; Jeong, Myung Ho; Song, Ho Cheon; Bom, Hee Seung [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2005-07-01

    The rapid donor cell death and rejection owing to humoral and cellular immune reactions are a basic limitation encountered in stem cell therapy for treatment of cardiovascular disease. We investigated the potential for longitudinal bioluminescence imaging to monitor the survival of transplanted stem cells prolonged by immunosuppressive agents. Embryonic rat H9c2 cardio myoblasts were transfected with adenovirus containing luciferase reporter gene (Ad-CMV-Fluc) in different MOI (1,10,100) and various cell doses (1x10{sup 5} - 5x10{sup 6})followed by injection in the thigh muscle of nude mice (n=6 per group), Other mice (n = 18) were undergone transient immunosuppression provided by either Cyclosporine (5mg/kg) or Tacrolimus (1mg/kg) or Dexamethasone (4mg/kg) beginning 3 days prior to and continuing to 2 weeks after transplantation. Optical bioluminescent imaging was then daily carried out using cooled CCD camera (Xenogen) Viral transfection at MOI 100 and the 5x10{sup 6} cell dose implantation resulted in optimal transgene efficiency. Mice received immunosuppressive agents displayed long-term in vivo reporter gene expression for a time course of 14 days. Tacrolimus (Prograf) and Cyclosporine successfully suppressed the transplanted cell loss in animals, that obviously observed until day 8 as compared to Dexamethasone-treated and non-treated mice (day 1: 1.00E+08 (Prograf), 9.47E+07 (Cys), 5.25E+07 (Dex), and 1.25E+07 p/s/cm{sup 2}/sr (control); day 8: 3.27E+05 (Prograf), 1.02E+05 (Cys), 6.17E+04 (Dex) and 2.73E+04 p/s/cm{sup 2}/sr (control)) and continued expressing bioluminescence until day 13 ( 6.42E+05 (Prograf), 4.99E+05 (Cys), and 4.10E+04 p/s/cm{sup 2}/sr. Induction of immune tolerance using pharmaceutical agents during cardio myoblast transplantation improved long-term donor cell survival in murine muscles. Optical imaging technique is capable of being used for tracking implanted stem cells in myocardium of living subjects over time.

  12. Bioluminescence imaging to monitor the prolongation of stem cell survival by pharmaceutical intervention

    International Nuclear Information System (INIS)

    The rapid donor cell death and rejection owing to humoral and cellular immune reactions are a basic limitation encountered in stem cell therapy for treatment of cardiovascular disease. We investigated the potential for longitudinal bioluminescence imaging to monitor the survival of transplanted stem cells prolonged by immunosuppressive agents. Embryonic rat H9c2 cardio myoblasts were transfected with adenovirus containing luciferase reporter gene (Ad-CMV-Fluc) in different MOI (1,10,100) and various cell doses (1x105 - 5x106)followed by injection in the thigh muscle of nude mice (n=6 per group), Other mice (n = 18) were undergone transient immunosuppression provided by either Cyclosporine (5mg/kg) or Tacrolimus (1mg/kg) or Dexamethasone (4mg/kg) beginning 3 days prior to and continuing to 2 weeks after transplantation. Optical bioluminescent imaging was then daily carried out using cooled CCD camera (Xenogen) Viral transfection at MOI 100 and the 5x106 cell dose implantation resulted in optimal transgene efficiency. Mice received immunosuppressive agents displayed long-term in vivo reporter gene expression for a time course of 14 days. Tacrolimus (Prograf) and Cyclosporine successfully suppressed the transplanted cell loss in animals, that obviously observed until day 8 as compared to Dexamethasone-treated and non-treated mice (day 1: 1.00E+08 (Prograf), 9.47E+07 (Cys), 5.25E+07 (Dex), and 1.25E+07 p/s/cm2/sr (control); day 8: 3.27E+05 (Prograf), 1.02E+05 (Cys), 6.17E+04 (Dex) and 2.73E+04 p/s/cm2/sr (control)) and continued expressing bioluminescence until day 13 ( 6.42E+05 (Prograf), 4.99E+05 (Cys), and 4.10E+04 p/s/cm2/sr. Induction of immune tolerance using pharmaceutical agents during cardio myoblast transplantation improved long-term donor cell survival in murine muscles. Optical imaging technique is capable of being used for tracking implanted stem cells in myocardium of living subjects over time

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

    Science.gov (United States)

    Davis, Matthew P; Sparks, John S; Smith, W Leo

    2016-01-01

    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.

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

    Science.gov (United States)

    Davis, Matthew P; Sparks, John S; Smith, W Leo

    2016-01-01

    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. PMID:27276229

  15. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes

    Science.gov (United States)

    Davis, Matthew P.; Sparks, John S.; Smith, W. Leo

    2016-01-01

    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. PMID:27276229

  16. Analytical Applications of Bioluminescence and Chemiluminescence

    Science.gov (United States)

    Chappelle, E. W. (Editor); Picciolo, G. L. (Editor)

    1975-01-01

    Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered.

  17. MEASUREMENTS OF BIOLUMINESCENCE IN DEEP SEA

    OpenAIRE

    Chikawa, M.; Kitamura, T; Nakagawa, Nakagawa; Yamamoto, I.; Wada, T.; Okei, K; Yamashita, Y.

    1996-01-01

    [Abstract] We have designed and built a photon counting system which measures low intensities of bioluminescence in deep sea. The system comprises a CCD-TV camera, two-dimensional image intensifier and video cassette recorder. Using this system we measured the vertical profile of bioluminescence in situ at the Suruga Trough and Nankai Trough to a depth of 3600 m and analyzed cultivated them.

  18. Noninvasive Bioluminescence Imaging in Small Animals

    OpenAIRE

    Zinn, Kurt R.; Chaudhuri, Tandra R.; Szafran, April Adams; O’Quinn, Darrell; Weaver, Casey; Dugger, Kari; Lamar, Dale; Kesterson, Robert A.; Wang, Xiangdong; Frank, Stuart J.

    2008-01-01

    There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase...

  19. Bioluminescent bioreporter assays for targeted detection of chemical and biological agents

    Science.gov (United States)

    Ripp, Steven; Jegier, Pat; Johnson, Courtney; Moser, Scott; Islam, Syed; Sayler, Gary

    2008-04-01

    Bioluminescent bioreporters carrying the bacterial lux gene cassette have been well established for the sensing and monitoring of select chemical agents. Their ability to generate target specific visible light signals with no requirement for extraneous additions of substrate or other hands-on manipulations affords a real-time, repetitive assaying technique that is remarkable in its simplicity and accuracy. Although the predominant application of lux-based bioluminescent bioreporters has been towards chemical compound detection, novel genetic engineering schemes are yielding a variety of new bioreporter systems that extend the lux sensing mechanism beyond mere analyte discrimination. For example, the unique specificity of bacteriophage (bacterial viruses) has been exploited in lux bioluminescent assays for specific identification of foodborne bacterial pathogens such as Escherichia coli O157:H7. With the concurrent ability to interface bioluminescent bioreporter assays onto integrated circuit microluminometers (BBICs; bioluminescent bioreporter integrated circuits), the potential exists for the development of sentinel microchips that can function as environmental monitors for multiplexed recognition of chemical and biological agents in air, food, and water. The size and portability of BBIC biosensors may ultimately provide a deployable, interactive network sensing technology adaptable towards chem/bio defense.

  20. Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch-activated ion channels.

    Science.gov (United States)

    Jin, Kelly; Klima, Jason C; Deane, Grant; Dale Stokes, Malcolm; Latz, Michael I

    2013-08-01

    Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.

  1. Noninvasive bioluminescence imaging in small animals.

    Science.gov (United States)

    Zinn, Kurt R; Chaudhuri, Tandra R; Szafran, April Adams; O'Quinn, Darrell; Weaver, Casey; Dugger, Kari; Lamar, Dale; Kesterson, Robert A; Wang, Xiangdong; Frank, Stuart J

    2008-01-01

    There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

  2. High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

    Science.gov (United States)

    Shultzaberger, Ryan K; Paddock, Mark L; Katsuki, Takeo; Greenspan, Ralph J; Golden, Susan S

    2015-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

  3. High throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

    Science.gov (United States)

    Shultzaberger, Ryan K.; Paddock, Mark L.; Katsuki, Takeo; Greenspan, Ralph J.; Golden, Susan S.

    2016-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise, and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

  4. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

    Science.gov (United States)

    England, Christopher G; Ehlerding, Emily B; Cai, Weibo

    2016-05-18

    The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

  5. High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

    Science.gov (United States)

    Shultzaberger, Ryan K; Paddock, Mark L; Katsuki, Takeo; Greenspan, Ralph J; Golden, Susan S

    2015-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

  6. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

    Science.gov (United States)

    England, Christopher G; Ehlerding, Emily B; Cai, Weibo

    2016-05-18

    The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

  7. Bioluminescence microscopy using a short focal-length imaging lens

    OpenAIRE

    Ogoh, K; Akiyoshi, R; May-Maw-Thet,; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H.

    2014-01-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera....

  8. Bacteria bioluminescent activity as an indicator of geomagnetic disturbances

    International Nuclear Information System (INIS)

    The effect of geomagnetic disturbances and storms on bioluminescence activity of bacterium were investigated. The bioluminescence intensity change depended on amplitude and continuous of geomagnetic storms. It is assumed, that the synchronization of luminous radiation take place in cellos when frequency of geomagnetic disturbances approached to an intrinsic one of a bioluminescence system. High sensitivity of bioluminescence of geomagnetic storms was detected. 5 refs., 4 figs

  9. Fluorescence and bioluminescence of bacterial luciferase intermediates

    International Nuclear Information System (INIS)

    An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at --200, was postulated to be an oxygenated reduced flavine-luciferase. Maintained and studied at --20 to --300, this material exhibits a relatively weak fluorescence emission peaking at about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming this fluorescence is lost and the fluorescence of flavine mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence. (auth)

  10. Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

    Science.gov (United States)

    Jiang, Tianyu; Wang, Weishan; Wu, Xingkang; Wu, Wenxiao; Bai, Haixiu; Ma, Zhao; Shen, Yuemao; Yang, Keqian; Li, Minyong

    2016-08-01

    In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system. PMID:26896339

  11. Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

    Science.gov (United States)

    Jiang, Tianyu; Wang, Weishan; Wu, Xingkang; Wu, Wenxiao; Bai, Haixiu; Ma, Zhao; Shen, Yuemao; Yang, Keqian; Li, Minyong

    2016-08-01

    In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.

  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. 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

    2014-12-01

    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 C(15)H(10)O(5), 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.

  14. 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

    2014-12-01

    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 C(15)H(10)O(5), 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

  15. Theoretical tuning of the firefly bioluminescence spectra by the modification of oxyluciferin

    Science.gov (United States)

    Cheng, Yuan-Yuan; Zhu, Jia; Liu, Ya-Jun

    2014-01-01

    Extending the firefly bioluminescence is of practical significance for the improved visualization of living cells and the development of a multicolor reporter. Tuning the color of bioluminescence in fireflies mainly involves the modification of luciferase and luciferin. In this Letter, we theoretically studied the emission spectra of 9 firefly oxyluciferin analogs in the gas phase and in solutions. Three density functionals, including B3LYP, CAM-B3LYP and M06-2X, were employed to theoretically predict the efficiently luminescent analogs. The reliable functionals for calculating the targeted systems were suggested. The luminescence efficiency, solvent effects, and substituent effects are discussed based on the calculated results.

  16. Inhibitory effect of lipoic acid on firefly luciferase bioluminescence

    International Nuclear Information System (INIS)

    Lipoic acid was found to inhibit the firefly luciferin-luciferase reaction. The inhibition is competitive and is the strongest known (Ki 0.026 ± 0.013 μM) compared with other reported inhibitors. Considering the structure-activity correlations, the mechanism of inhibition may originate from the sulfur atom and carboxyl moiety of lipoic acid giving it structural specificity. Subsequent addition of lipoic acid and nitric oxide accelerated the inhibition in vitro, suggesting that lipoic acid may have a functional role in regulating firefly bioluminescence

  17. In vivo cell tracking with bioluminescence imaging.

    Science.gov (United States)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol

    2015-03-01

    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.

  18. Optimisation of acquisition time in bioluminescence imaging

    Science.gov (United States)

    Taylor, Shelley L.; Mason, Suzannah K. G.; Glinton, Sophie; Cobbold, Mark; Styles, Iain B.; Dehghani, Hamid

    2015-03-01

    Decreasing the acquisition time in bioluminescence imaging (BLI) and bioluminescence tomography (BLT) will enable animals to be imaged within the window of stable emission of the bioluminescent source, a higher imaging throughput and minimisation of the time which an animal is anaesthetised. This work investigates, through simulation using a heterogeneous mouse model, two methods of decreasing acquisition time: 1. Imaging at fewer wavelengths (a reduction from five to three); and 2. Increasing the bandwidth of filters used for imaging. The results indicate that both methods are viable ways of decreasing the acquisition time without a loss in quantitative accuracy. Importantly, when choosing imaging wavelengths, the spectral attenuation of tissue and emission spectrum of the source must be considered, in order to choose wavelengths at which a high signal can be achieved. Additionally, when increasing the bandwidth of the filters used for imaging, the bandwidth must be accounted for in the reconstruction algorithm.

  19. 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.

  20. In vivo cell tracking with bioluminescence imaging

    International Nuclear Information System (INIS)

    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

  1. A Multichannel Bioluminescence Determination Platform for Bioassays.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi

    2016-01-01

    The present protocol introduces a multichannel bioluminescence determination platform allowing a high sample throughput determination of weak bioluminescence with reduced standard deviations. The platform is designed to carry a multichannel conveyer, an optical filter, and a mirror cap. The platform enables us to near-simultaneously determine ligands in multiple samples without the replacement of the sample tubes. Furthermore, the optical filters beneath the multichannel conveyer are designed to easily discriminate colors during assays. This optical system provides excellent time- and labor-efficiency to users during bioassays.

  2. A Multichannel Bioluminescence Determination Platform for Bioassays.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi

    2016-01-01

    The present protocol introduces a multichannel bioluminescence determination platform allowing a high sample throughput determination of weak bioluminescence with reduced standard deviations. The platform is designed to carry a multichannel conveyer, an optical filter, and a mirror cap. The platform enables us to near-simultaneously determine ligands in multiple samples without the replacement of the sample tubes. Furthermore, the optical filters beneath the multichannel conveyer are designed to easily discriminate colors during assays. This optical system provides excellent time- and labor-efficiency to users during bioassays. PMID:27424912

  3. Chemistry and biology of insect bioluminescence

    International Nuclear Information System (INIS)

    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)

  4. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    Science.gov (United States)

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  5. Noninvasive visualization of microRNA-16 in the chemoresistance of gastric cancer using a dual reporter gene imaging system.

    Directory of Open Access Journals (Sweden)

    Fu Wang

    Full Text Available MicroRNAs (miRNAs have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS and firefly luciferase (Fluc genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of (131I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo (99mTc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16 and 5-fluorouracil (5-FU activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.

  6. Bioluminescence for determining energy state of plants

    Science.gov (United States)

    Ching, T. M.

    1975-01-01

    Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered.

  7. 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.

  8. Bioluminescence: diplôme 2015

    OpenAIRE

    Giachino, Vincent; Geiser, Martial

    2016-01-01

    Dans le cadre du projet européen BRAAVOO, qui vise à mesurer les concentrations de différents polluants présents dans la mer, nous développons un lecteur de bioluminescence embarqué dans une bouée.

  9. Bioluminescent bioreporter integrated circuit detection methods

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

    2005-06-14

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for detection of particular analytes, including ammonia and estrogen compounds.

  10. Bubble stimulation efficiency of dinoflagellate bioluminescence.

    Science.gov (United States)

    Deane, Grant B; Stokes, M Dale; Latz, Michael I

    2016-02-01

    Dinoflagellate bioluminescence, a common source of bioluminescence in coastal waters, is stimulated by flow agitation. Although bubbles are anecdotally known to be stimulatory, the process has never been experimentally investigated. This study quantified the flash response of the bioluminescent dinoflagellate Lingulodinium polyedrum to stimulation by bubbles rising through still seawater. Cells were stimulated by isolated bubbles of 0.3-3 mm radii rising at their terminal velocity, and also by bubble clouds containing bubbles of 0.06-10 mm radii for different air flow rates. Stimulation efficiency, the proportion of cells producing a flash within the volume of water swept out by a rising bubble, decreased with decreasing bubble radius for radii less than approximately 1 mm. Bubbles smaller than a critical radius in the range 0.275-0.325 mm did not stimulate a flash response. The fraction of cells stimulated by bubble clouds was proportional to the volume of air in the bubble cloud, with lower stimulation levels observed for clouds with smaller bubbles. An empirical model for bubble cloud stimulation based on the isolated bubble observations successfully reproduced the observed stimulation by bubble clouds for low air flow rates. High air flow rates stimulated more light emission than expected, presumably because of additional fluid shear stress associated with collective buoyancy effects generated by the high air fraction bubble cloud. These results are relevant to bioluminescence stimulation by bubbles in two-phase flows, such as in ship wakes, breaking waves, and sparged bioreactors.

  11. Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence

    Science.gov (United States)

    Virta, Marko; Lineri, Sanna; Kankaanpää, Pasi; Karp, Matti; Peltonen, Karita; Nuutila, Jari; Lilius, Esa-Matti

    1998-01-01

    Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples. PMID:9464386

  12. Enhanced Landweber algorithm via Bregman iterations for bioluminescence tomography

    Science.gov (United States)

    Xia, Yi; Zhang, Meng

    2014-09-01

    Bioluminescence tomography (BLT) is an important optical molecular imaging modality aimed at visualizing physiological and pathological processes at cellular and molecular levels. While the forward process of light propagation is described by the diffusion approximation to radiative transfer equation, BLT is the inverse problem to reconstruct the 3D localization and quantification of internal bioluminescent sources distribution. Due to the inherent ill-posedness of the BLT problem, regularization is generally indispensable to obtain more favorable reconstruction. In particular, total variation (TV) regularization is known to be effective for piecewise-constant source distribution which can permit sharp discontinuities and preserve edges. However, total variation regularization generally suffers from the unsatisfactory staircasing effect. In this work, we introduce the Bregman iterative regularization to alleviate this degeneration and enhance the numerical reconstruction of BLT. Based on the existing Landweber method (LM), we put forward the Bregman-LM-TV algorithm for BLT. Numerical experiments are carried out and preliminary simulation results are reported to evaluate the proposed algorithms. It is found that Bregman-LM-TV can significantly outperform the individual Landweber method for BLT when the source distribution is piecewise-constant.

  13. Bioluminescence microscopy using a short focal-length imaging lens.

    Science.gov (United States)

    Ogoh, K; Akiyoshi, R; May-Maw-Thet; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H

    2014-03-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

  14. Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

    Science.gov (United States)

    Kheirabadi, Mitra; Sharafian, Zohreh; Naderi-Manesh, Hossein; Heineman, Udo; Gohlke, Ulrich; Hosseinkhani, Saman

    2013-12-01

    Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

  15. Reliability of a bioluminescence ATP assay for detection of bacteria.

    OpenAIRE

    Selan, L.; Berlutti, F; Passariello, C.; Thaller, M C; Renzini, G

    1992-01-01

    The reliability of bioluminescence assays which employ the luciferin-luciferase ATP-dependent reaction to evaluate bacterial counts was studied, both in vitro and on urine specimens. Bioluminescence and cultural results for the most common urinary tract pathogens were analyzed. Furthermore, the influence of the culture medium, of the assaying method, and of the phase of growth on bioluminescence readings was studied. Results show that Proteus, Providencia, and Morganella strains are not corre...

  16. Bioluminescent Probe for Detecting Mercury(II) in Living Mice.

    Science.gov (United States)

    Jiang, Tianyu; Ke, Bowen; Chen, Hui; Wang, Weishan; Du, Lupei; Yang, Keqian; Li, Minyong

    2016-08-01

    A novel bioluminescence probe for mercury(II) was obtained on the basis of the distinct deprotection reaction of dithioacetal to decanal, so as to display suitable sensitivity and selectivity toward mercury(II) over other ions with bacterial bioluminescence signal. These experimental results indicated such a probe was a novel promising method for mercury(II) bioluminescence imaging in environmental and life sciences ex vivo and in vivo. PMID:27412583

  17. Spectrally resolved bioluminescence tomography using the reciprocity approach

    OpenAIRE

    Dehghani, Hamid; Davis, Scott C.; Pogue, Brian W.

    2008-01-01

    Spectrally resolved bioluminescence optical tomography is an approach to recover images of, for example, Luciferase activity within a volume using multiwavelength emission data from internal bioluminescence sources. The underlying problem of uniqueness associated with nonspectrally resolved intensity-based bioluminescence tomography is demonstrated and it is shown that using a non-negative constraint inverse algorithm, an accurate solution for the source distribution can be calculated from th...

  18. Effect of electromagnetic fields on the bacteria bioluminescent activity

    International Nuclear Information System (INIS)

    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

  19. Bioluminescence in the high Arctic during the polar night

    OpenAIRE

    Berge, Jørgen; Båtnes, Anna Solvang; Johnsen, Geir; Blackwell, Susan; Moline, Mark A.

    2012-01-01

    This study examines the composition and activity of the planktonic community during the polar night in the high Arctic Kongsfjord, Svalbard. Our results are the first published evidence of bioluminescence among zooplankton during the Arctic polar night. The observations were collected by a bathyphotometer detecting bioluminescence, integrated into an autonomous underwater vehicle, to determine the concentration and intensity of bioluminescent flashes as a function of time of day and depth. To...

  20. Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging.

    Science.gov (United States)

    Ayzenberg, Ilya; Schlevogt, Sibylle; Metzdorf, Judith; Stahlke, Sarah; Pedreitturia, Xiomara; Hunfeld, Anika; Couillard-Despres, Sebastien; Kleiter, Ingo

    2015-01-01

    Bioluminescence imaging is a sensitive approach for longitudinal neuroimaging. Transgenic mice expressing luciferase under the promoter of doublecortin (DCX-luc), a specific marker of neuronal progenitor cells (NPC), allow monitoring of neurogenesis in living mice. Since the extent and time course of neurogenesis during autoimmune brain inflammation are controversial, we investigated neurogenesis in MOG-peptide induced experimental allergic encephalomyelitis (EAE) using DCX-luc reporter mice. We observed a marked, 2- to 4-fold increase of the bioluminescence signal intensity 10 days after EAE induction and a gradual decline 1-2 weeks thereafter. In contrast, immunostaining for DCX revealed no differences between EAE and control mice 2 and 4 weeks after immunization in zones of adult murine neurogenesis such as the dentate gyrus. Ex vivo bioluminescence imaging showed similar luciferase expression in brain homogenates of EAE and control animals. Apart from complete immunization including MOG-peptide also incomplete immunization with complete Freund´s adjuvant and pertussis toxin resulted in a rapid increase of the in vivo bioluminescence signal. Blood-brain barrier (BBB) leakage was demonstrated 10 days after both complete and incomplete immunization and might explain the increased bioluminescence signal in vivo. We conclude, that acute autoimmune inflammation in EAE does not alter neurogenesis, at least at the stage of DCX-expressing NPC. Effects of immunization on the BBB integrity must be considered when luciferase is used as a reporter within the CNS during the active stage of EAE. Models with stable CNS-restricted luciferase expression could serve as technically convenient way to evaluate BBB integrity in a longitudinal manner.

  1. Cloning and characterization of new bioluminescent proteins

    Science.gov (United States)

    Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce

    1999-07-01

    Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

  2. 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.

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

    International Nuclear Information System (INIS)

    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/cm2. 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.

  4. Understanding Bioluminescence in Dinoflagellates—How Far Have We Come?

    Science.gov (United States)

    Valiadi, Martha; Iglesias-Rodriguez, Debora

    2013-01-01

    Some dinoflagellates possess the remarkable genetic, biochemical, and cellular machinery to produce bioluminescence. Bioluminescent species appear to be ubiquitous in surface waters globally and include numerous cosmopolitan and harmful taxa. Nevertheless, bioluminescence remains an enigmatic topic in biology, particularly with regard to the organisms’ lifestyle. In this paper, we review the literature on the cellular mechanisms, molecular evolution, diversity, and ecology of bioluminescence in dinoflagellates, highlighting significant discoveries of the last quarter of a century. We identify significant gaps in our knowledge and conflicting information and propose some important research questions that need to be addressed to advance this research field.

  5. Human reporter genes: potential use in clinical studies

    International Nuclear Information System (INIS)

    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

  6. A transcriptional and proteomic survey of Arachnocampa luminosa (Diptera: Keroplatidae) lanterns gives insights into the origin of bioluminescence from the Malpighian tubules in Diptera.

    Science.gov (United States)

    Silva, J R; Amaral, D T; Hastings, J W; Wilson, T; Viviani, V R

    2015-11-01

    Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera.

  7. Phylogenetic and transcriptomic analyses reveal the evolution of bioluminescence and light detection in marine deep-sea shrimps of the family Oplophoridae (Crustacea: Decapoda).

    Science.gov (United States)

    Wong, Juliet M; Pérez-Moreno, Jorge L; Chan, Tin-Yam; Frank, Tamara M; Bracken-Grissom, Heather D

    2015-02-01

    Bioluminescence is essential to the survival of many organisms, particularly in the deep sea where light is limited. Shrimp of the family Oplophoridae exhibit a remarkable mechanism of bioluminescence in the form of a secretion used for predatory defense. Three of the ten genera possess an additional mode of bioluminescence in the form of light-emitting organs called photophores. Phylogenetic analyses can be useful for tracing the evolution of bioluminescence, however, the few studies that have attempted to reconcile the relationships within Oplophoridae have generated trees with low-resolution. We present the most comprehensive phylogeny of Oplophoridae to date, with 90% genera coverage using seven genes (mitochondrial and nuclear) across 30 oplophorid species. We use our resulting topology to trace the evolution of bioluminescence within Oplophoridae. Previous studies have suggested that oplophorid visual systems may be tuned to differentiate the separate modes of bioluminescence. While all oplophorid shrimp possess a visual pigment sensitive to blue-green light, only those bearing photophores have an additional pigment sensitive to near-ultraviolet light. We attempt to characterize opsins, visual pigment proteins essential to light detection, in two photophore-bearing species (Systellaspis debilis and Oplophorus gracilirostris) and make inferences regarding their function and evolutionary significance.

  8. Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter.

    Science.gov (United States)

    Lopes, Nicholas; Hawkins, Shawn A; Jegier, Patricia; Menn, Fu-Min; Sayler, Gary S; Ripp, Steven

    2012-01-01

    The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCM( lux ), was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.

  9. Development of bacteriophage-based bioluminescent bioreporters for monitoring of microbial pathogens

    Science.gov (United States)

    Ozen, Aysu; Montgomery, Kacey; Jegier, Pat; Patterson, Stacey; Daumer, Kathleen A.; Ripp, Steven A.; Garland, Jay L.; Sayler, Gary S.

    2004-03-01

    Microorganisms pose numerous problems when present in human occupied enclosed environments. Primary among these are health related hazards, manifested as infectious diseases related to contaminated drinking water, food, or air circulation systems or non-infectious allergy related complications associated with microbial metabolites (sick building syndrome). As a means towards rapid detection of microbial pathogens, we are attempting to harness the specificity of bacterial phage for their host with a modified quorum sensing amplification signal to produce quantifiable bioluminescent (lux) detection on a silicon microluminometer. The bacteriophage itself is metabolically inactive, only achieving replicative capabilities upon infection of its specific host bacterium. Bacteriophage bioluminescent bioreporters contain a genomically inserted luxI component. During an infection event, the phage genes and accompanying luxI construct are taken up by the host bacterium and transcribed, resulting in luxI expression and subsequent activation of a homoserine lactone inducible bioluminescent bioreporter. We constructed a vector carrying the luxI gene under the control of a strong E. coli promoter and cloned it into E. coli. We have shown that it can induce luminescence up to 14,000 counts per second when combined with the bioreporter strain. In their final embodiment, these sensors will be fully independent microelectronic monitors for microbial contamination, requiring only exposure of the biochip to the sample, with on-chip signal processing downloaded directly to the local area network of the environmental control system.

  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

    Full Text Available BACKGROUND: 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. PRINCIPAL FINDINGS: 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. SIGNIFICANCE: 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

  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 REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    Science.gov (United States)

    This review of the recent literature on environmental applications of bioluminescence systems will focus on in vivo and in vitro bioluminescence methods that have been utilized to elucidate properties of chemicals, toxic and mutagenic effects, and to estimate biomass. The unifyin...

  13. Detection of ATP and NADH: A Bioluminescent Experience.

    Science.gov (United States)

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  14. Use of the liquid scintillation spectrometer in bioluminescence analysis

    International Nuclear Information System (INIS)

    This review covers publications concerning analytical bioluminescence which in the main have appeared between mid-1973 and mid-1976. Outlines of some new assays and techniques are given together with modifications of existing procedures. Comments are presented on the use of the liquid scintillation spectrometer and other equipment for measuring bioluminescence. New applications are detailed and discussed

  15. 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.

  16. Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

    Science.gov (United States)

    Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

    2013-08-01

    Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

  17. Dinoflagellate bioluminescence in response to mechanical stimuli in water flows

    Directory of Open Access Journals (Sweden)

    A. S. Cussatlegras

    2005-01-01

    Full Text Available Bioluminescence of plankton organisms induced by water movements has long been observed and is still under investigations because of its great complexity. In particular, the exact mechanism occurring at the level of the cell has not been yet fully understood. This work is devoted to the study of the bioluminescence of the dinoflagellates plankton species Pyrocystis noctiluca in response to mechanical stimuli generated by water flows. Several experiments were performed with different types of flows in a Couette shearing apparatus. All of them converge to the conclusion that stationary homogeneous laminar shear does not trigger massive bioluminescence, but that acceleration and shear are both necessary to stimulate together an intense bioluminescence response. The distribution of the experimental bioluminescence thresholds is finally calculated from the light emission response for the Pyrocystis noctiluca species.

  18. The bottom oxygen border of bioluminescence distribution in ocean

    Science.gov (United States)

    Zavoruev, Valerii

    2006-02-01

    From materials of forwarding researches follows, that the depth of deposition of the bottom border of bioluminescence plankton has not correlation with any of measurable hydrological parameters. As the reaction of bioluminescence is oxygen depended, it was logical to assume, that the situation of the bottom maximum of luminescence plankton is determined by concentration of oxygen. The data of vertical distribution of bioluminescence intensity of plankton and concentration of oxygen received in the Black Sea and near to east coast of America were investigated. Is established, that the deep maximum of bioluminescence of plankton is found out between isooxygen 0.35 and 0.20 ml/l. At concentration of oxygen in water is lower 0.10-0.20 ml/l the bioluminescence of plankton it is not found out.

  19. Bioluminescence in the high Arctic during the polar night.

    Science.gov (United States)

    Berge, J; Båtnes, A S; Johnsen, G; Blackwell, S M; Moline, M A

    2012-01-01

    This study examines the composition and activity of the planktonic community during the polar night in the high Arctic Kongsfjord, Svalbard. Our results are the first published evidence of bioluminescence among zooplankton during the Arctic polar night. The observations were collected by a bathyphotometer detecting bioluminescence, integrated into an autonomous underwater vehicle, to determine the concentration and intensity of bioluminescent flashes as a function of time of day and depth. To further understand community dynamics and composition, plankton nets were used to collect organisms passing through the bathyphotometer along with traditional vertical net tows. Additionally, using a moored bathyphotometer closed to the sampling site, the bioluminescence potential itself was shown not to have a diurnal or circadian rhythm. Rather, our results provide evidence for a diel vertical migration of bioluminescent zooplankton that does not correspond to any externally detectable changes in illumination.

  20. Dinoflagellate bioluminescence in response to mechanical stimuli in water flows

    Science.gov (United States)

    Cussatlegras, A. S.; Le Gal, P.

    2005-02-01

    Bioluminescence of plankton organisms induced by water movements has long been observed and is still under investigations because of its great complexity. In particular, the exact mechanism occurring at the level of the cell has not been yet fully understood. This work is devoted to the study of the bioluminescence of the dinoflagellates plankton species Pyrocystis noctiluca in response to mechanical stimuli generated by water flows. Several experiments were performed with different types of flows in a Couette shearing apparatus. All of them converge to the conclusion that stationary homogeneous laminar shear does not trigger massive bioluminescence, but that acceleration and shear are both necessary to stimulate together an intense bioluminescence response. The distribution of the experimental bioluminescence thresholds is finally calculated from the light emission response for the Pyrocystis noctiluca species.

  1. Moment searching algorithm for bioluminescence tomography

    Institute of Scientific and Technical Information of China (English)

    Ludong Jin; Yan Wu; Jie Tian; Heyu Huang; Xiaochao Qu

    2009-01-01

    To avoid the ill-posedness in the inverse problem of bioluminescence tomography, a moment searching algorithm fusing the finite element method (FEM) with the moment concept in theoretical mechanics is developed. In the algorithm, the source's information is mapped to the surface photon flux density by FEM, and the source's position is modified with the feedback through the algorithm of barycenter searching, which makes full use of the position information of the photon flux density on surface. The position is modified in every iterative step and will finally converge to the real source's value theoretically.

  2. Bioluminescence imaging of wave-induced turbulence

    Science.gov (United States)

    Stokes, M. Dale; Deane, Grant B.; Latz, Michael I.; Rohr, Jim

    2004-01-01

    The ability to measure turbulent processes on small spatial and temporal scales is a long standing problem in physical oceanography. Here we explore a novel means of measuring fluid shear stress using the cell flashing behavior of bioluminescent dinoflagellates. To illustrate this technique, we present estimates of the heterogeneous, time-varying shear stress inside a breaking wave crest. These results have implications for a better understanding of upper ocean wave physics, air-sea gas transfer, and the biology of planktonic near-surface organisms as well as providing a new quantitative fluid visualization tool.

  3. A causal relation between bioluminescence and oxygen to quantify the cell niche

    OpenAIRE

    Lambrechts, Dennis; Roeffaers, Maarten; Goossens, Karel; Hofkens, Johan; Vande Velde, Greetje; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-01-01

    Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for ce...

  4. Molecular phylogeny of Squaliformes and first occurrence of bioluminescence in sharks

    OpenAIRE

    Straube, N.; Li, C; Claes, J.M.; Corrigan, S.; Naylor, G

    2015-01-01

    Background Squaliform sharks represent approximately 27 % of extant shark diversity, comprising more than 130 species with a predominantly deep-dwelling lifestyle. Many Squaliform species are highly specialized, including some that are bioluminescent, a character that is reported exclusively from Squaliform sharks within Chondrichthyes. The interfamiliar relationships within the order are still not satisfactorily resolved. Herein we estimate the phylogenetic interrelationships of a generic le...

  5. Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

    Science.gov (United States)

    Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C; Merk, Denis R; Lyons, Jennifer K; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N; Ray, Pritha; Patel, Manishkumar; Chang, Ya-Fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C; Dash, Rajesh; Yang, Phillip C; Brinton, Todd J; Yock, Paul G; McConnell, Michael V; Gambhir, Sanjiv S

    2016-09-01

    Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article. PMID:27308957

  6. Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44.

    Science.gov (United States)

    Chauhan, Archana; Layton, Alice C; Williams, Daniel E; Smartt, Abby E; Ripp, Steven; Karpinets, Tatiana V; Brown, Steven D; Sayler, Gary S

    2011-09-01

    Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids.

  7. Draft Genome Sequence of the Polycyclic Aromatic Hydrocarbon-Degrading, Genetically Engineered Bioluminescent Bioreporter Pseudomonas fluorescens HK44

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, Archana [ORNL; Layton, Alice [University of Tennessee, Knoxville (UTK); Williams, Daniel W [ORNL; Smart, Abby E. [University of Tennessee, Knoxville (UTK); Ripp, Steven Anthony [ORNL; Karpinets, Tatiana V [ORNL; Brown, Steven D [ORNL; Sayler, Gary Steven [ORNL

    2011-01-01

    Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of {approx}6.1 Mb sequence indicates that 30% of the traits are unique and distributed over 5 genomic islands, a prophage and two plasmids.

  8. 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.

  9. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    Science.gov (United States)

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

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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)

  12. Multimodal imaging of orthotopic hepatocellular carcinoma using small animal PET, bioluminescence and contrast enhanced CT imaging

    International Nuclear Information System (INIS)

    Molecular imaging with small-animal PET and bioluminescence imaging has been used as an important tool in cancer research. One of the disadvantages of these imaging modalities is the lack of anatomic information. To obtain fusion images with both molecular and anatomical information, small-animal PET and bioluminescence images fused with contrast enhance CT image in orthotopic hepatocellular carcinoma (HCC) model. We retrovially transfected dual gene (HSV1-tk and firefly luciferase) to morris hepatoma cells. The expression of HSV1-tk and luciferase was checked by optical imager and in vitro radiolabeled FIAU uptake, respectively and also checked by RT-PCR analysis. MCA-TL cells (5X105/ 0.05 ml) mixed with matrigel (1: 10) injected into left lobe of liver in nude mice. 124I-FIAU-PET, bioluminescence and contrast enhanced CT images were obtained in the orthotopic HCC model and digital whole body autoradiography (DWBA) was performed. Small animal PET image was obtained at 2 h post injection of 124I-FIAU and contrast enhanced CT image was obtained at 3 h post injection of Fenestra LC (0.3 ml). MCA-TL cells showed more specific 124I-FIAU uptake and higher luminescent activity than parental cells. The orthotopic HCC was detected by 124I-FIAU PET, contrast enhanced CT, and BLI and confirmed by DWBA. Registered image in orthotopic HCC t models showed a good correlation of images from both PET and CT. Contrast enhanced CT image delineated margin of HCC. Multimodal imaging with 124I-FIAU PET, bioluminescence and contrast enhanced CT allows a precise and improved detection of tumor in orthotopic hepatocellular carcinoma model. Multimodal imaging is potentially useful for monitoring progression of hepatic metastasis and for the evaluation of cancer treatments

  13. A bioluminescent assay for measuring glucose uptake.

    Science.gov (United States)

    Valley, Michael P; Karassina, Natasha; Aoyama, Natsuyo; Carlson, Coby; Cali, James J; Vidugiriene, Jolanta

    2016-07-15

    Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts. PMID:27130501

  14. Development of bioluminescent Salmonella strains for use in food safety

    Directory of Open Access Journals (Sweden)

    Bailey R Hartford

    2008-01-01

    Full Text Available Abstract Background Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, we developed bioluminescent Salmonella strains that can be used for real-time monitoring of the pathogen's growth on food products. To accomplish this, twelve Salmonella strains from the broiler production continuum were transformed with the broad host range plasmid pAKlux1, and a chicken skin attachment model was developed. Results Salmonella strains carrying pAKlux1 constitutively expressed the luxCDABE operon and were therefore detectable using bioluminescence. Strains were characterized in terms of bioluminescence properties and plasmid stability. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, we developed an attachment model using chicken skin. The effect of washing on attachment of Salmonella strains to chicken skin was tested using bioluminescent strains, which revealed the attachment properties of each strain. Conclusion This study demonstrated that bioluminescence is a sensitive and effective tool to detect Salmonella on food products in real-time. Bioluminescence imaging is a promising technology that can be utilized to evaluate new food safety measures for reducing Salmonella contamination on food products.

  15. 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

  16. Fimbrolide Natural Products Disrupt Bioluminescence of Vibrio By Targeting Autoinducer Biosynthesis and Luciferase Activity.

    Science.gov (United States)

    Zhao, Weining; Lorenz, Nicola; Jung, Kirsten; Sieber, Stephan A

    2016-01-18

    Vibrio is a model organism for the study of quorum sensing (QS) signaling and is used to identify QS-interfering drugs. Naturally occurring fimbrolides are important tool compounds known to affect QS in various organisms; however, their cellular targets have so far remained elusive. Here we identify the irreversible fimbrolide targets in the proteome of living V. harveyi and V. campbellii via quantitative mass spectrometry utilizing customized probes. Among the major hits are two protein targets with essential roles in Vibrio QS and bioluminescence. LuxS, responsible for autoinducer 2 biosynthesis, and LuxE, a subunit of the luciferase complex, were both covalently modified at their active-site cysteines leading to inhibition of activity. The identification of LuxE unifies previous reports suggesting inhibition of bioluminescence downstream of the signaling cascade and thus contributes to a better mechanistic understanding of these QS tool compounds.

  17. Measuring IL-1β Processing by Bioluminescence Sensors I: Using a Bioluminescence Resonance Energy Transfer Biosensor.

    Science.gov (United States)

    Compan, Vincent; Pelegrín, Pablo

    2016-01-01

    IL-1β processing is one of the hallmarks of inflammasome activation and drives the initiation of the inflammatory response. For decades, Western blot or ELISA have been extensively used to study this inflammatory event. Here, we describe the use of a bioluminescence resonance energy transfer (BRET) biosensor to monitor IL-1β processing in real time and in living macrophages either using a plate reader or a microscope.

  18. Bioluminescence-activated deep-tissue photodynamic therapy of cancer.

    Science.gov (United States)

    Kim, Yi Rang; Kim, Seonghoon; Choi, Jin Woo; Choi, Sung Yong; Lee, Sang-Hee; Kim, Homin; Hahn, Sei Kwang; Koh, Gou Young; Yun, Seok Hyun

    2015-01-01

    Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm(2) for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT.

  19. Bioluminescence: a versatile technique for imaging cellular and molecular features

    Science.gov (United States)

    Paley, Miranda A.

    2016-01-01

    Bioluminescence is a ubiquitous imaging modality for visualizing biological processes in vivo. This technique employs visible light and interfaces readily with most cell and tissue types, making it a versatile technology for preclinical studies. Here we review basic bioluminescence imaging principles, along with applications of the technology that are relevant to the medicinal chemistry community. These include noninvasive cell tracking experiments, analyses of protein function, and methods to visualize small molecule metabolites. In each section, we also discuss how bioluminescent tools have revealed insights into experimental therapies and aided drug discovery. Last, we highlight the development of new bioluminescent tools that will enable more sensitive and multi-component imaging experiments and, thus, expand our broader understanding of living systems.

  20. Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

    Science.gov (United States)

    Modestova, Yulia; Koksharov, Mikhail I; Ugarova, Natalia N

    2014-09-01

    Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.

  1. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    Science.gov (United States)

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  2. The terrestrial bioluminescent animals of Japan.

    Science.gov (United States)

    Oba, Yuichi; Branham, Marc A; Fukatsu, Takema

    2011-11-01

    Light production by organisms, or bioluminescence, has fascinated not only scientists but also ordinary people all over the world, and it has been especially so in Japan. Here we review the biological information available to date for all luminous terrestrial animals known from Japan, particularly focusing on their diversity and systematics, their biology and ecology in Japan, and putative function and biochemistry of their luminescence. In total 58 luminous terrestrial animals have been described from Japan, which consist of 50 fireflies (Coleoptera: Lampyridae), one glowworm beetle (Coleoptera: Phengodidae), two fungus gnats (Diptera: Keroplatidae), one springtail (Collembola), one millipede (Diplopoda), one centipede (Chilopoda) and two earthworms (Oligochaeta). For all except some firefly species, the DNA "barcode" sequences of a cytochrome oxidase subunit I region are provided. We also introduce how intricately the seasonal appearance and glimmering of luminous insects, in particular those of fireflies, have been interwoven into the culture, art, literature and mentality of Japanese people. PMID:22035300

  3. 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....

  4. Dinoflagellate bioluminescence in response to mechanical stimuli in water flows

    OpenAIRE

    Cussatlegras, A. S.; Gal, P.

    2005-01-01

    International audience Bioluminescence of plankton organisms induced by water movements has long been observed and is still under investigations because of its great complexity. In particular, the exact mechanism occurring at the level of the cell has not been yet fully understood. This work is devoted to the study of the bioluminescence of the dinoflagellates plankton species Pyrocystis noctiluca in response to mechanical stimuli generated by water flows. Several experiments were performe...

  5. Bioluminescence-Sensing Assay for Microbial Growth Recognition

    OpenAIRE

    Heba Ramadan Eed; Abdel-Kader, Nora S.; Mahmoud Helmy El Tahan; Tianhong Dai; Rehab Amin

    2016-01-01

    The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP) bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A biolumine...

  6. Real-Time Bioluminescence Imaging of Nitroreductase in Mouse Model.

    Science.gov (United States)

    Feng, Ping; Zhang, Huateng; Deng, Quankun; Liu, Wei; Yang, Linghui; Li, Guobo; Chen, Guo; Du, Lupei; Ke, Bowen; Li, Minyong

    2016-06-01

    Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors; however, its precise detection in living cells and animals remains a considerable challenge. Herein, we developed three reaction-based probes and a related bioluminescence assay for the real-time NTR detection. The high sensitivity and selectivity of probe 3, combined with its remarkable potential of bioluminescence imaging, affords a valuable approach for in vivo imaging of NTR in a tumor model mouse. PMID:27197544

  7. Real-Time Bioluminescence Imaging of Nitroreductase in Mouse Model.

    Science.gov (United States)

    Feng, Ping; Zhang, Huateng; Deng, Quankun; Liu, Wei; Yang, Linghui; Li, Guobo; Chen, Guo; Du, Lupei; Ke, Bowen; Li, Minyong

    2016-06-01

    Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors; however, its precise detection in living cells and animals remains a considerable challenge. Herein, we developed three reaction-based probes and a related bioluminescence assay for the real-time NTR detection. The high sensitivity and selectivity of probe 3, combined with its remarkable potential of bioluminescence imaging, affords a valuable approach for in vivo imaging of NTR in a tumor model mouse.

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

    International Nuclear Information System (INIS)

    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

  9. Bioluminescence in dinoflagellates – diversity, molecular phylogeny and field ecology

    OpenAIRE

    Valiadi, Martha

    2011-01-01

    Marine dinoflagellates are an ecologically important group of protists within the plankton, performing key process such as photosynthesis, heterotrophy and toxin production. Some dinoflagellates are also capable of producing bioluminescence and they are the most abundant protists that produce light in the surface waters of the oceans. This study employed molecular tools to investigate the identity of bioluminescent species, the genetic basis, diversity and functional regulation of biolumin...

  10. Effect of SNPs in protein kinase Czgene on gene expression in the reporter gene detection system

    Institute of Scientific and Technical Information of China (English)

    Zhuo Liu; Hong-Xia Sun; Yong-Wei Zhang; Yun-Feng Li; Jin Zuo; Yan Meng; Fu-De Fang

    2004-01-01

    AIM: To investigated the effects of the SNPs (rs411021,rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes.METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above.These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity of the luciferase was assayed. Effects of the SNPs on RNA splicing were analyzed by bioinformatics.RESULTS: rs427811T allele and rs809912G allele enhanced the activity of the reporter gene expression. None of the 5 SNPs affected RNA splicing.CONCLUSION: SNPs in protein kinase Cz (PKCZ) gene probably play a role in the susceptibility to type 2 diabetes by affecting the expression level of the relevant genes.

  11. Interactive graphic editing tools in bioluminescent imaging simulation

    Science.gov (United States)

    Li, Hui; Tian, Jie; Luo, Jie; Wang, Ge; Cong, Wenxiang

    2005-04-01

    It is a challenging task to accurately describe complicated biological tissues and bioluminescent sources in bioluminescent imaging simulation. Several graphic editing tools have been developed to efficiently model each part of the bioluminescent simulation environment and to interactively correct or improve the initial models of anatomical structures or bioluminescent sources. There are two major types of graphic editing tools: non-interactive tools and interactive tools. Geometric building blocks (i.e. regular geometric graphics and superquadrics) are applied as non-interactive tools. To a certain extent, complicated anatomical structures and bioluminescent sources can be approximately modeled by combining a sufficient large number of geometric building blocks with Boolean operators. However, those models are too simple to describe the local features and fine changes in 2D/3D irregular contours. Therefore, interactive graphic editing tools have been developed to facilitate the local modifications of any initial surface model. With initial models composed of geometric building blocks, interactive spline mode is applied to conveniently perform dragging and compressing operations on 2D/3D local surface of biological tissues and bioluminescent sources inside the region/volume of interest. Several applications of the interactive graphic editing tools will be presented in this article.

  12. Stimulation of bioluminescence in Noctiluca sp. using controlled temperature changes.

    Science.gov (United States)

    Han, Jing; Li, GuiJuan; Liu, HuanYing; Hu, HaoHao; Zhang, XueGang

    2013-01-01

    Bioluminescence induced by multifarious stimuli has long been observed and is remains under investigation because of its great complexity. In particular, the exact mechanism underlying bioluminescence is not yet fully understood. This work presents a new experimental method for studying Noctiluca sp. bioluminescence under temperature change stimulation. It is a study of Noctiluca sp. bioluminescence using controlled temperature changes in a tank. A characteristic of this experiment is the large volume of water used (1 m(3) in a tank of 2 × 1 × 1 m). Temperature changes were controlled by two methods. In the first, a flask filled with hot water was introduced into the tank and in the second, a water heater was used in the tank. Temperature changes were recorded using sensors. Noctiluca sp. bioluminescence was recorded using a Canon 5D Mark II and this allowed the characteristics of Noctiluca sp. bioluminescence under temperature change stimulation to be monitored.

  13. In Vivo Bioluminescent Imaging (BLI: Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

    Directory of Open Access Journals (Sweden)

    Steven Ripp

    2010-12-01

    Full Text Available In vivo bioluminescent imaging (BLI is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism.

  14. EVALUATION OF MONKEYPOX VIRUS INFECTION OF BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS) USING IN VIVO BIOLUMINESCENT IMAGING

    OpenAIRE

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

    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 no...

  15. 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

  16. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke. PMID:27038754

  17. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

  18. Bioluminescence tracking of alginate micro-encapsulated cell transplants.

    Science.gov (United States)

    Tiernan, Aubrey R; Sambanis, Athanassios

    2014-07-22

    Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba(2+) crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere.

    OpenAIRE

    Kragelund, L.; Hosbond, C; Nybroe, O

    1997-01-01

    The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain resp...

  20. aequorine bioluminescence response to calcium in vitro and in cerebral cortex

    OpenAIRE

    Tricoire, Ludovic

    2006-01-01

    During my PhD, I investigated in vitro the calcium-dependent bioluminescence of thephotoprotein aequorin and then used its bioluminescence to image neuronal activities in theneocortical network. This genetically encoded calcium sensor can be expressed in specific cell types and its bioluminescence is not toxic and exhibit a high signal/noise ratio.I first search for mutations modifying aequorin bioluminescence, using a randommutagenesis and in vitro evolution approach. I isolated mutants show...

  1. Modelling dinoflagellates as an approach to the seasonal forecasting of bioluminescence in the North Atlantic

    OpenAIRE

    Marcinko, Charlotte L.J.; Martin, Adrian P.; Allen, John T.

    2014-01-01

    Bioluminescence within ocean surface waters is of significant interest because it can enhance the study of subsurface movement and organisms. Little is known about how bioluminescence potential (BPOT) varies spatially and temporally in the open ocean. However, light emitted from dinoflagellates often dominates the stimulated bioluminescence field. As a first step towards forecasting surface ocean bioluminescence in the open ocean, a simple ecological model is developed which simulates seasona...

  2. Vegetable seed radiosensitivity and kinetic analysis of super-weak bioluminescence

    International Nuclear Information System (INIS)

    Bioluminescence of several vegetable seeds induced by γ-rays was studied. The results show that positive relation exists between seeds bioluminescence and irradiation dose, which fits with equation Y=Y0eKD. The higher the K value is, the more intense the bioluminescence induced by γ-rays is. Significant differences among K values were found with different varieties. The bioluminescence and exterior measurement value of seed radiosensitivity showed good consistency

  3. In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model

    Directory of Open Access Journals (Sweden)

    Shane eMassey

    2011-08-01

    Full Text Available Bioluminescent imaging (BLI technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5x103 bacteria and monitored by BLI at 24, 48 and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria.

  4. Click beetle luciferases as dual reporters of gene expression in Candida albicans.

    Science.gov (United States)

    Kapitan, Mario; Eichhof, Isabel; Lagadec, Quentin; Ernst, Joachim F

    2016-08-01

    Synthetic genes encoding functional luciferases of the click beetle (CB) Pyrophorus plagiophthalamus have been expressed in the human fungal pathogen Candida albicans. Both green- and red-emitting CB luciferases (CaCBGluc and CaCBRluc) were produced with high efficiency in transformants under transcriptional control of the growth-dependent ACT1 promoter, as well as by the HWP1 and UME6 promoters, which are upregulated during hyphal morphogenesis, as well as by the YWP1 and EFG1 promoters, which are downregulated. For all hyphally regulated genes, relative bioluminescence values derived from promoter fusions approximated relative transcript levels of native genes, although downregulation of YWP1 promoter activity required correction for the stability of CB luciferases (approximate half-lives 30 min for CaCBRluc and 80 min for CaCBGluc, as determined by immunoblotting). Importantly, the activity of both luciferases could be separately monitored in a single strain, in intact cells, in lysed cells or in cell extracts using luciferin as single substrate and inhibition of hypha formation by farnesol could be easily detected by the HWP1p-CaCBRluc fusion. The results suggest that CB luciferases are convenient tools to measure gene expression in C. albicans and may facilitate screenings for antifungal compounds. PMID:27339610

  5. 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

  6. Rapid detection (4 h) of methicillin-resistant Staphylococcus aureus by a bioluminescence method.

    OpenAIRE

    Park, C. H.; Hixon, D L; McLaughlin, C M; Cook, J F

    1988-01-01

    A 4-h bioluminescence method for methicillin susceptibility determination was compared with reference methods. Of the Staphylococcus aureus strains tested, 80 were methicillin resistant, 180 were methicillin susceptible, and 10 were borderline susceptible. There was 100% correlation between bioluminescence and reference methods for methicillin-susceptible and methicillin-resistant strains. All borderline-susceptible strains were identified as methicillin resistant by bioluminescence.

  7. Novel bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution for Renilla luciferase.

    Science.gov (United States)

    Jiang, Tianyu; Yang, Xiaofeng; Yang, Xingye; Yuan, Mingliang; Zhang, Tianchao; Zhang, Huateng; Li, Minyong

    2016-06-21

    Two series of novel coelenterazine analogues (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution have been designed and synthesized successfully for the extension of bioluminescent substrates. After extensive evaluation, some compounds display excellent bioluminescence properties compared with DeepBlueC in cellulo, thus becoming potential molecules for bioluminescence techniques. PMID:27197767

  8. A bioluminescent mouse model of proliferation to highlight early stages of pancreatic cancer: A suitable tool for preclinical studies.

    Science.gov (United States)

    de Latouliere, Luisa; Manni, Isabella; Iacobini, Carla; Pugliese, Giuseppe; Grazi, Gian Luca; Perri, Pasquale; Cappello, Paola; Novelli, Franco; Menini, Stefano; Piaggio, Giulia

    2016-09-01

    Transgenic mouse models designed to recapitulate genetic and pathologic aspects of cancer are useful to study early stages of disease as well as its progression. Among several, two of the most sophisticated models for pancreatic ductal adenocarcinoma (PDAC) are the LSL-Kras(G12D/+);Pdx-1-Cre (KC) and LSL-Kras(G12D/+);LSL-Trp53(R172H/+);Pdx-1-Cre (KPC) mice, in which the Cre-recombinase regulated by a pancreas-specific promoter activates the expression of oncogenic Kras alone or in combination with a mutant p53, respectively. Non-invasive in vivo imaging offers a novel approach to preclinical studies introducing the possibility to investigate biological events in the spatio/temporal dimension. We recently developed a mouse model, MITO-Luc, engineered to express the luciferase reporter gene in cells undergoing active proliferation. In this model, proliferation events can be visualized non-invasively by bioluminescence imaging (BLI) in every body district in vivo. Here, we describe the development and characterization of MITO-Luc-KC- and -KPC mice. In these mice we have now the opportunity to follow PDAC evolution in the living animal in a time frame process. Moreover, by relating in vivo and ex vivo BLI and histopathological data we provide evidence that these mice could represents a suitable tool for pancreatic cancer preclinical studies. Our data also suggest that aberrant proliferation events take place early in pancreatic carcinogenesis, before tumour appearance. PMID:26704357

  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. PMID:20540359

  10. Quantum/molecular mechanics study of firefly bioluminescence on luciferase oxidative conformation

    Science.gov (United States)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-07-01

    This is the first report of a computational study of the color tuning mechanism of firefly bioluminescence, using the oxidative conformation of luciferase. The results of these calculations demonstrated that the electrostatic field generated by luciferase is fundamental both for the emission shift and efficiency. Further calculations indicated that a shift in emission is achieved by modulating the energy, at different degrees, of the emissive and ground states. These differences in energy modulation will then lead to changes in the energy gap between the states.

  11. Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

    Science.gov (United States)

    Ronald, John A.; Cusso, Lorena; Chuang, Hui-Yen; Yan, Xinrui; Dragulescu-Andrasi, Anca; Gambhir, Sanjiv Sam

    2013-01-01

    Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×106 cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at

  12. The Expanding Toolbox of In Vivo Bioluminescent Imaging

    Science.gov (United States)

    Xu, Tingting; Close, Dan; Handagama, Winode; Marr, Enolia; Sayler, Gary; Ripp, Steven

    2016-01-01

    In vivo bioluminescent imaging (BLI) permits the visualization of engineered bioluminescence from living cells and tissues to provide a unique perspective toward the understanding of biological processes as they occur within the framework of an authentic in vivo environment. The toolbox of in vivo BLI includes an inventory of luciferase compounds capable of generating bioluminescent light signals along with sophisticated and powerful instrumentation designed to detect and quantify these light signals non-invasively as they emit from the living subject. The information acquired reveals the dynamics of a wide range of biological functions that play key roles in the physiological and pathological control of disease and its therapeutic management. This mini review provides an overview of the tools and applications central to the evolution of in vivo BLI as a core technology in the preclinical imaging disciplines. PMID:27446798

  13. The Expanding Toolbox of In Vivo Bioluminescent Imaging.

    Science.gov (United States)

    Xu, Tingting; Close, Dan; Handagama, Winode; Marr, Enolia; Sayler, Gary; Ripp, Steven

    2016-01-01

    In vivo bioluminescent imaging (BLI) permits the visualization of engineered bioluminescence from living cells and tissues to provide a unique perspective toward the understanding of biological processes as they occur within the framework of an authentic in vivo environment. The toolbox of in vivo BLI includes an inventory of luciferase compounds capable of generating bioluminescent light signals along with sophisticated and powerful instrumentation designed to detect and quantify these light signals non-invasively as they emit from the living subject. The information acquired reveals the dynamics of a wide range of biological functions that play key roles in the physiological and pathological control of disease and its therapeutic management. This mini review provides an overview of the tools and applications central to the evolution of in vivo BLI as a core technology in the preclinical imaging disciplines. PMID:27446798

  14. Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit

    Science.gov (United States)

    Bolton, Eric K.; Sayler, Gary S.; Nivens, David E.; Rochelle, James M.; Ripp, Steven; Simpson, Michael L.

    2002-01-01

    We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells. c2002 Elsevier Science B.V. All rights reserved.

  15. Space application research of EMCCDs for bioluminescence imaging

    Science.gov (United States)

    Zhang, Tao

    The detection of bioluminescense is widely used on the ground, while the detection of bioluminescence in space is still at the stage of detecting bright bioluminescense. With the rapid development of research in Space Life Sciences, it will be necessary to develop a detection technology to detect weak bioluminescense. Compared to other low-light detection techniques for ground, there are more advantages of EMCCDs for space application. Build a space bioluminescence imaging detection system, analysis the feasibility and capability of its will be significant. Co-Author:Xie Zongbao,Zheng Weibo

  16. Effect of irradiation on bioluminescence spectrum of microbial ATP

    International Nuclear Information System (INIS)

    The effect of irradiation on bioluminescence spectrum of dehydrated cabbage microbial ATP was studied. The results showed that the spectral bandwidth of ATP standard was from 490 to 640 nm and the peak wavelength was at 563 nm. The spectral bandwidths of irradiated dehydrated cabbage microbial ATP and CK did not change. Peak wavelengths of dehydrated cabbage irradiated at different dosages were not significantly different from that of CK. The peaks of bioluminescence spectrum of irradiated samples were higher than that of CK, which may be because of the increasing concentration of ATP, and this effect would be kept for quite a long time after irradiation. (authors)

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

    Science.gov (United States)

    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 Ca(2+)-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 1.30Å 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 (473-474nm with no shoulder at 400nm). Fluorescence spectra of Ca(2+)-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca(2+)-discharged aequorin, but different from Ca(2+)-discharged obelins and clytin which fluorescence is red-shifted by 25-30nm 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.

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

    Science.gov (United States)

    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 Ca(2+)-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 1.30Å 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 (473-474nm with no shoulder at 400nm). Fluorescence spectra of Ca(2+)-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca(2+)-discharged aequorin, but different from Ca(2+)-discharged obelins and clytin which fluorescence is red-shifted by 25-30nm 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. PMID:27395792

  19. 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.

  20. Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

    Directory of Open Access Journals (Sweden)

    Ludmila A. Frank

    2010-12-01

    Full Text Available Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

  1. 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. PMID:24518318

  2. 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.

  3. Molecular phylogeny and node time estimation of bioluminescent Lantern Sharks (Elasmobranchii: Etmopteridae).

    Science.gov (United States)

    Straube, Nicolas; Iglésias, Samuel P; Sellos, Daniel Y; Kriwet, Jürgen; Schliewen, Ulrich K

    2010-09-01

    Deep-sea Lantern Sharks (Etmopteridae) represent the most speciose family within Dogfish Sharks (Squaliformes). We compiled an extensive DNA dataset to estimate the first molecular phylogeny of the family and to provide node age estimates for the origin and diversification for this enigmatic group. Phylogenetic inferences yielded consistent and well supported hypotheses based on 4685bp of both nuclear (RAG1) and mitochondrial genes (COI, 12S-partial 16S, tRNAVal and tRNAPhe). The monophyletic family Etmopteridae originated in the early Paleocene around the C/T boundary, and split further into four morphologically distinct lineages supporting three of the four extant genera. The exception is Etmopterus which is paraphyletic with respect to Miroscyllium. Subsequent rapid radiation within Etmopterus in the Oligocene/early Miocene was accompanied by divergent evolution of bioluminescent flank markings which morphologically characterize the four lineages. Higher squaliform interrelationships could not be satisfactorily identified, but convergent evolution of bioluminescence in Dalatiidae and Etmopteridae is supported.

  4. Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).

    Science.gov (United States)

    Amaral, Danilo T; Mitani, Yasuo; Ohmiya, Yoshihiro; Viviani, Vadim R

    2016-07-25

    Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis. PMID:27060405

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

    International Nuclear Information System (INIS)

    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/cm2/sr) from the knee joint transplanted with cells approximately increased 10-fold more than (9.43E+07p/s/cm2/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

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

    Institute of Scientific and Technical Information of China (English)

    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 ki-nase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-β-D- arabi-nofuranosyl-5-iodouracil (131I-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 131I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1×109, 5×108, 1×108, 5×107 and 1×107 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1×109, 5×108, 1×108, 5×107, 1×107 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131I-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 tran-scription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSV1-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 131I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images qual-ity 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 5×107 pfu of virus titer. The result could be set better in 1~5×108 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 HSV1-tk/131I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter gene imaging

  7. Filtering and deconvolution for bioluminescence imaging of small animals

    International Nuclear Information System (INIS)

    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)

  8. The influence of SHFEMF on bioluminescence of V. Harveyi

    International Nuclear Information System (INIS)

    Exposure of bacteria V. harveyi grown on agar medium to 7 HHz electromagnetic field changes the intensity of their luminescence. It is suggested that the dynamics of the luminescence change reflects the adaptation processes in the microorganisms which accompany the electromao.netic field effect. The changes observed may be attributed to the temperature dependence of bioluminescence

  9. The mechanism of electronic excitation in the bacterial bioluminescent reaction

    International Nuclear Information System (INIS)

    The current state of the problem of formation of the electron-excited product in the chemiluminescent reaction that underlies the bacterial luminescence is analysed. Various schemes of chemical transformations capable of producing a bacterial bioluminescence emitter are presented. The problem of excitation of secondary emitters is considered; two possible mechanisms of their excitation are analysed.

  10. Photoacoustic microscopy of tyrosinase reporter gene in vivo

    OpenAIRE

    Krumholz, Arie; VanVickle-Chavez, Sarah J.; Yao, Junjie; Fleming, Timothy P.; Gillanders, William E.; Wang, Lihong V.

    2011-01-01

    Photoacoustic tomography is a hybrid modality based on optical absorption excitation and ultrasonic detection. It is sensitive to melanin, one of the primary absorbers in skin. For cells that do not naturally contain melanin, melanin production can be induced by introducing the gene for tyrosinase, the primary enzyme responsible for expression of melanin in melanogenic cells. Optical resolution photoacoustic microscopy was used in the ex vivo study reported here, where the signal from transfe...

  11. Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds.

    Science.gov (United States)

    Yamamoto, Ryoko; Takegami, Shigehiko; Konishi, Atsuko; Horikawa, Hikari; Yonezawa, Sayumi; Kitade, Tatsuya

    2016-06-01

    In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 μmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 μmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes. PMID:27146598

  12. Boosting bioluminescence neuroimaging: an optimized protocol for brain studies.

    Science.gov (United States)

    Aswendt, Markus; Adamczak, Joanna; Couillard-Despres, Sebastien; Hoehn, Mathias

    2013-01-01

    Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (i.v., i.p., s.c.), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000-300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.

  13. Bioluminescent imaging of HPV-positive oral tumor growth and its response to image-guided radiotherapy.

    Science.gov (United States)

    Zhong, Rong; Pytynia, Matt; Pelizzari, Charles; Spiotto, Michael

    2014-04-01

    The treatment paradigms for head and neck squamous cell cancer (HNSCC) are changing due to the emergence of human papillomavirus (HPV)-associated tumors possessing distinct molecular profiles and responses to therapy. Although patients with HNSCCs are often treated with radiotherapy, preclinical models are limited by the ability to deliver precise radiation to orthotopic tumors and to monitor treatment responses accordingly. To better model this clinical scenario, we developed a novel autochthonous HPV-positive oral tumor model to track responses to small molecules and image-guided radiation. We used a tamoxifen-regulated Cre recombinase system to conditionally express the HPV oncogenes E6 and E7 as well as a luciferase reporter (iHPV-Luc) in the epithelial cells of transgenic mice. In the presence of activated Cre recombinase, luciferase activity, and by proxy, HPV oncogenes were induced to 11-fold higher levels. In triple transgenic mice containing the iHPV-Luc, K14-CreER(tam), and LSL-Kras transgenes, tamoxifen treatment resulted in oral tumor development with increased bioluminescent activity within 6 days that reached a maximum of 74.8-fold higher bioluminescence compared with uninduced mice. Oral tumors expressed p16 and MCM7, two biomarkers associated with HPV-positive tumors. After treatment with rapamycin or image-guided radiotherapy, tumors regressed and possessed decreased bioluminescence. Thus, this novel system enables us to rapidly visualize HPV-positive tumor growth to model existing and new interventions using clinically relevant drugs and radiotherapy techniques.

  14. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA

    OpenAIRE

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-01-01

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratical...

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

    Energy Technology Data Exchange (ETDEWEB)

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    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.

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

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable 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.

  17. Effect of concentrating and exposing the bioluminescent bacteria to the non-luminescent allo-bacterial extracellular products on their luminescence

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, J.; Priya, G.G.; Kannapiran, E.

    Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal...

  18. Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.

    Science.gov (United States)

    Kim, Jiho; Grailhe, Regis

    2016-08-01

    Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry. PMID:27144967

  19. Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.

    Science.gov (United States)

    Kim, Jiho; Grailhe, Regis

    2016-08-01

    Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.

  20. In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.

    Directory of Open Access Journals (Sweden)

    Jean-René Martin

    Full Text Available Many different cells' signalling pathways are universally regulated by Ca(2+ concentration [Ca(2+] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+ reporter GFP-aequorin (GA in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+] transients in the Mushroom Bodies (MBs following nicotine stimulation were accompanied by a delayed secondary [Ca(2+] rise (up to 15 min. later in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain.

  1. In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.

    Science.gov (United States)

    Martin, Jean-René; Rogers, Kelly L; Chagneau, Carine; Brûlet, Philippe

    2007-03-07

    Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.

  2. In vivo Bioluminescence Imaging of Ca2+ Signalling in the Brain of Drosophila

    Science.gov (United States)

    Chagneau, Carine; Brûlet, Philippe

    2007-01-01

    Many different cells' signalling pathways are universally regulated by Ca2+ concentration [Ca2+] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca2+ reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca2+] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca2+] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca2+] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain. PMID:17342209

  3. Quantification of bioluminescence images of point source objects using diffusion theory models

    International Nuclear Information System (INIS)

    A simple approach for estimating the location and power of a bioluminescent point source inside tissue is reported. The strategy consists of using a diffuse reflectance image at the emission wavelength to determine the optical properties of the tissue. Following this, bioluminescence images are modelled using a single point source and the optical properties from the reflectance image, and the depth and power are iteratively adjusted to find the best agreement with the experimental image. The forward models for light propagation are based on the diffusion approximation, with appropriate boundary conditions. The method was tested using Monte Carlo simulations, Intralipid tissue-simulating phantoms and ex vivo chicken muscle. Monte Carlo data showed that depth could be recovered within 6% for depth 4-12 mm, and the corresponding relative source power within 12%. In Intralipid, the depth could be estimated within 8% for depth 4-12 mm, and the relative source power, within 20%. For ex vivo tissue samples, source depths of 4.5 and 10 mm and their relative powers were correctly identified

  4. Potential of the FES-hERL PET reporter gene system - Basic evaluation for gene therapy monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Furukawa, Takako [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan)]. E-mail: takakof@fmsrsa.fukui-med.ac.jp; Lohith, Talakad G. [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Takamatsu, Shinji [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Tanaka, Takeshi [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Department of Otorhinolaryngology, University of Fukui, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan)

    2006-01-15

    Purpose: In vivo reporter genes can be powerful tools in supporting and ensuring the success of gene therapy. A careful and rational design of a reporter system is essential to realize a noninvasive in vivo reporter gene imaging system applicable for humans. We designed a new in vivo reporter gene imaging system that uses F-18-labeled estradiol (FES) and human estrogen receptor ligand (hERL) binding domain, taking advantage that FES is a radiopharmaceutical already being used for human studies with access to a wide range of tissues, including the brain, and that hERL lacking DNA binding domain can no longer work as a transcription factor, and carried out basic studies to evaluate its potential for gene therapy monitoring. Methods: We constructed a plasmid (pTIER) to coexpress a model therapeutic gene and the reporter gene hERL and transfected Cos7 cells and examined their uptake of [{sup 3}H]estradiol and FES in culture media. The uptake of FES by mouse calf muscle electroporated with pTIER was also tested. Results: The cells transfected with pTIER took up the radioligands efficiently and specifically in culture media. Also, the mouse calf muscle electroporated with pTIER accumulated a higher amount of FES than did the control. Conclusion: The data indicate that our new reporter gene system seems promising for in vivo imaging of gene expression and gene therapy monitoring.

  5. Modelling dinoflagellates as an approach to the seasonal forecasting of bioluminescence in the North Atlantic

    Science.gov (United States)

    Marcinko, Charlotte L. J.; Martin, Adrian P.; Allen, John T.

    2014-11-01

    Bioluminescence within ocean surface waters is of significant interest because it can enhance the study of subsurface movement and organisms. Little is known about how bioluminescence potential (BPOT) varies spatially and temporally in the open ocean. However, light emitted from dinoflagellates often dominates the stimulated bioluminescence field. As a first step towards forecasting surface ocean bioluminescence in the open ocean, a simple ecological model is developed which simulates seasonal changes in dinoflagellate abundance. How forecasting seasonal changes in BPOT may be achieved through combining such a model with relationships derived from observations is discussed and an example is given. The study illustrates a potential new approach to forecasting BPOT through explicitly modelling the population dynamics of a prolific bioluminescent phylum. The model developed here offers a promising platform for the future operational forecasting of the broad temporal changes in bioluminescence within the North Atlantic. Such forecasting of seasonal patterns could provide valuable information for the targeting of scientific field campaigns.

  6. Recovering 3D tumor locations from 2D bioluminescence images and registration with CT images

    Science.gov (United States)

    Huang, Xiaolei; Metaxas, Dimitris N.; Menon, Lata G.; Mayer-Kuckuk, Philipp; Bertino, Joseph R.; Banerjee, Debabrata

    2006-02-01

    In this paper, we introduce a novel and efficient algorithm for reconstructing the 3D locations of tumor sites from a set of 2D bioluminescence images which are taken by a same camera but after continually rotating the object by a small angle. Our approach requires a much simpler set up than those using multiple cameras, and the algorithmic steps in our framework are efficient and robust enough to facilitate its use in analyzing the repeated imaging of a same animal transplanted with gene marked cells. In order to visualize in 3D the structure of the tumor, we also co-register the BLI-reconstructed crude structure with detailed anatomical structure extracted from high-resolution microCT on a single platform. We present our method using both phantom studies and real studies on small animals.

  7. Impact of Anesthesia Protocols on In Vivo Bioluminescent Bacteria Imaging Results

    OpenAIRE

    Chuzel, Thomas; Sanchez, Violette; Vandamme, Marc; Martin, Stéphane; Flety, Odile; Pager, Aurélie; Chabanel, Christophe; Magnier, Luc; Foskolos, Marie; Petit, Océane; Rokbi, Bachra; Chereul, Emmanuel

    2015-01-01

    Infectious murine models greatly benefit from optical imaging using bioluminescent bacteria to non-invasively and repeatedly follow in vivo bacterial infection. In this context, one of the most critical parameters is the bioluminescence sensitivity to reliably detect the smallest number of bacteria. Another critical point is the anesthetic approaches that have been demonstrated to impact the bioluminescence flux emission in studies with luciferase-transfected tumor cells. However, this impact...

  8. A study on bioluminescence and photoluminescence in the earthworm Eisenia lucens.

    Science.gov (United States)

    Pes, O; Midlik, A; Schlaghamersky, J; Zitnan, M; Taborsky, P

    2016-02-01

    Eisenia lucens is an earthworm living in the organic soil layer of decomposing wood. When irritated, the worm expels coelomic fluid through pores in its body wall, exhibiting blue-green bioluminescence. The mechanism of the bioluminescence, which seems to be different from other bioluminescence systems of terrestrial animals, has been studied in this work. Many lines of evidence indicate that riboflavin stored in coelomycetes plays an important role in this glowing reaction.

  9. Phage-amplified bioluminescent bioreporters for the detection of foodborne pathogens

    Science.gov (United States)

    Ripp, Steven; Young, Jacque C.; Ozen, Aysu; Jegier, Patricia; Johnson, Courtney; Daumer, Kathleen; Garland, Jay; Sayler, Gary S.

    2004-06-01

    The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.

  10. Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

    Science.gov (United States)

    Jia, Kun; Ionescu, Rodica Elena

    2016-01-01

    : Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

  11. 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.

  12. 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.

  13. Bacterial bioluminescence and Gumbel statistics: From quorum sensing to correlation

    Science.gov (United States)

    Delle Side, Domenico; Velardi, Luciano; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Talà, Adelfia; Salvatore Tredici, Maurizio

    2013-12-01

    We show that, in particular experimental conditions, the time course of the radiant fluxes, measured from a bioluminescent emission of a Vibrio harveyi related strain, collapse after suitable rescaling onto the Gumbel distribution of extreme value theory. We argue that the activation times of the strain luminous emission follow the universal behavior described by this statistical law, in spite of the fact that no extremal process is known to occur.

  14. Bioluminescence imaging: a shining future for cardiac regeneration

    OpenAIRE

    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...

  15. Bioluminescence for USP sterility testing of pharmaceutical suspension products.

    OpenAIRE

    Bussey, D M; K. Tsuji

    1986-01-01

    Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was e...

  16. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    OpenAIRE

    Lv, Yujie; Tian, Jie; Cong, Wenxiang; Wang, Ge

    2007-01-01

    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 s...

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

    International Nuclear Information System (INIS)

    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

  18. Robust image modeling technique with a bioluminescence image segmentation application

    Science.gov (United States)

    Zhong, Jianghong; Wang, Ruiping; Tian, Jie

    2009-02-01

    A robust pattern classifier algorithm for the variable symmetric plane model, where the driving noise is a mixture of a Gaussian and an outlier process, is developed. The veracity and high-speed performance of the pattern recognition algorithm is proved. Bioluminescence tomography (BLT) has recently gained wide acceptance in the field of in vivo small animal molecular imaging. So that it is very important for BLT to how to acquire the highprecision region of interest in a bioluminescence image (BLI) in order to decrease loss of the customers because of inaccuracy in quantitative analysis. An algorithm in the mode is developed to improve operation speed, which estimates parameters and original image intensity simultaneously from the noise corrupted image derived from the BLT optical hardware system. The focus pixel value is obtained from the symmetric plane according to a more realistic assumption for the noise sequence in the restored image. The size of neighborhood is adaptive and small. What's more, the classifier function is base on the statistic features. If the qualifications for the classifier are satisfied, the focus pixel intensity is setup as the largest value in the neighborhood.Otherwise, it will be zeros.Finally,pseudo-color is added up to the result of the bioluminescence segmented image. The whole process has been implemented in our 2D BLT optical system platform and the model is proved.

  19. 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.

  20. Synchronization of circadian bioluminescence as a group-foraging strategy in cave glowworms.

    Science.gov (United States)

    Maynard, Andrew J; Merritt, David J

    2013-07-01

    Flies of the genus Arachnocampa are sit-and-lure predators that use bioluminescence to attract flying prey to their silk webs. Some species are most common in rainforest habitat and others inhabit both caves and rainforest. We have studied the circadian regulation of bioluminescence in two species: one found in subtropical rainforest with no known cave populations and the other found in temperate rainforest with large populations in limestone caves. The rainforest species is typical of most nocturnal animals in that individuals are entrained by the light:dark (LD) cycle to be active at night; in this case, their propensity to bioluminesce is greatest at night. The dual-habitat species shows an opposite phase response to the same entrainment; its bioluminescence propensity rhythm is entrained by LD exposure to peak during the day. Nevertheless, in LD environments, individuals do not bioluminesce during the day because ambient light inhibits their bioluminescence (negative masking), pushing bioluminescence into the dark period. This unusual and unexpected phenomenon could be related to their association with caves and has been suggested to be an adaptation of the circadian system that promotes synchronization of a colony's output of bioluminescence. Here, we use controlled laboratory experiments to show that individuals do synchronize their bioluminescence rhythms when in visual contact with each other. Entrainment of the bioluminescence rhythm to the biological photophase causes colony-wide synchronization, creating a daily sinusoidal rhythm of the intensity of bioluminescence in the many thousands of individuals making up a colony. This synchronization could provide a group-foraging advantage, allowing the colony to glow most brightly when the prey are most likely to be active. PMID:23575257

  1. Synchronization of circadian bioluminescence as a group-foraging strategy in cave glowworms.

    Science.gov (United States)

    Maynard, Andrew J; Merritt, David J

    2013-07-01

    Flies of the genus Arachnocampa are sit-and-lure predators that use bioluminescence to attract flying prey to their silk webs. Some species are most common in rainforest habitat and others inhabit both caves and rainforest. We have studied the circadian regulation of bioluminescence in two species: one found in subtropical rainforest with no known cave populations and the other found in temperate rainforest with large populations in limestone caves. The rainforest species is typical of most nocturnal animals in that individuals are entrained by the light:dark (LD) cycle to be active at night; in this case, their propensity to bioluminesce is greatest at night. The dual-habitat species shows an opposite phase response to the same entrainment; its bioluminescence propensity rhythm is entrained by LD exposure to peak during the day. Nevertheless, in LD environments, individuals do not bioluminesce during the day because ambient light inhibits their bioluminescence (negative masking), pushing bioluminescence into the dark period. This unusual and unexpected phenomenon could be related to their association with caves and has been suggested to be an adaptation of the circadian system that promotes synchronization of a colony's output of bioluminescence. Here, we use controlled laboratory experiments to show that individuals do synchronize their bioluminescence rhythms when in visual contact with each other. Entrainment of the bioluminescence rhythm to the biological photophase causes colony-wide synchronization, creating a daily sinusoidal rhythm of the intensity of bioluminescence in the many thousands of individuals making up a colony. This synchronization could provide a group-foraging advantage, allowing the colony to glow most brightly when the prey are most likely to be active.

  2. 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

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

    International Nuclear Information System (INIS)

    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 l2 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 rather

  4. la bioluminescence de l'aequorine en réponse au calcium In vitro et dans le Cortex cerebral

    OpenAIRE

    Tricoire, Ludovic

    2006-01-01

    During my PhD, I investigated in vitro the calcium-dependent bioluminescence of thephotoprotein aequorin and then used its bioluminescence to image neuronal activities in theneocortical network. This genetically encoded calcium sensor can be expressed in specific cell types and its bioluminescence is not toxic and exhibit a high signal/noise ratio.I first search for mutations modifying aequorin bioluminescence, using a randommutagenesis and in vitro evolution approach. I isolated mutants show...

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

    Science.gov (United States)

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

    2006-12-01

    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.

  6. An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

    Science.gov (United States)

    Fourrage, Cécile; Swann, Karl; Gonzalez Garcia, Jose Raul; Campbell, Anthony K; Houliston, Evelyn

    2014-04-09

    Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

  7. 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.

  8. Inflammatory modulating effects of low level laser therapy on iNOS expression by means of bioluminescence imaging

    Science.gov (United States)

    Moriyama, Yumi; Moriyama, Eduardo H.; Blackmore, Kristina; Akens, Margarete K.; Lilge, Lothar

    2005-09-01

    This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm-2, 5 J cm-2) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.

  9. Bacterial Bioluminescence: Spectral Study of the Emitters in the In Vivo Reaction

    NARCIS (Netherlands)

    Matheson, I.B.C.; Lee, J.; Muller, F.

    1981-01-01

    Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily dist

  10. The rapid bioluminescence assay method for content of bacteria in dehydrated vegetable and condiment before radiation

    International Nuclear Information System (INIS)

    The microbial colony-forming unit (cfu) in dehydrated vegetable and condiment was determined by using ATP bioluminescence method. The result showed that bioluminescence of ATP was correlative to the microbial cfu obviously. The detecting time was within 1-2 h. This method could be applied to determine micro load of products before irradiation sterilization. (authors)

  11. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    International Nuclear Information System (INIS)

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity

  12. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  13. Reporter Gene Assay for Detection of Shellfish Toxins

    Institute of Scientific and Technical Information of China (English)

    WEI-DONG YANG; MIN-YI WU; JIE-SHENG LIU; XI-CHUN PENG; HONG-YE LI

    2009-01-01

    Objective To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry. Methods A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software. Results Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX 2,3 from 0 to 16 ng/mL. Conclusion pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

  14. Visualization of the Biological Behavior of Tumor-Associated Macrophages in Living Mice with Colon Cancer Using Multimodal Optical Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Yun Ju Choi

    2016-03-01

    Full Text Available We sought to visualize the migration of tumor-associated macrophages (TAMs to tumor lesions and to evaluate the effects of anti-inflammatory drugs on TAM-modulated tumor progression in mice with colon cancer using a multimodal optical reporter gene system. Murine macrophage Raw264.7 cells expressing an enhanced firefly luciferase (Raw/effluc and murine colon cancer CT26 cells coexpressing Rluc and mCherry (CT26/Rluc-mCherry, CT26/RM were established. CT26/RM tumor-bearing mice received Raw/effluc via their tail veins, and combination of bioluminescence imaging (BLI and fluorescence imaging (FLI was conducted for in vivo imaging of TAMs migration and tumor progression. Dexamethasone (DEX, a potent anti-inflammatory drug, was administered intraperitoneally to tumor-bearing mice following the intravenous transfer of Raw/effluc cells. The migration of TAMs and tumor growth was monitored by serial FLI and BLI. The migration of Raw/effluc cells to tumor lesions was observed at day 1, and BLI signals were still distinct at tumor lesions on day 4. Localization of BLI signals from migrated Raw/effluc cells corresponded to that of FLI signals from CT26/RM tumors. In vivo FLI of tumors demonstrated enhanced tumor growth associated with macrophage migration to tumor lesions. Treatment with DEX inhibited the influx of Raw/effluc cells to tumor lesions and abolished the enhanced tumor growth associated with macrophage migration. These findings suggest that molecular imaging approach for TAM tracking is a valuable tool for evaluating the role of TAMs in the tumor microenvironment as well as for the development of new drugs to control TAM involvement in the modulation of tumor progression.

  15. Preliminary Report of Molecular Detection of Retinoblastoma Gene Mutations

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    To develop gene diagnosis for retinoblastoma predisposition, it is necessary to disclose the retinoblastoma gene mutations or deletions in detail. Genomic DNA from tumor and peripheral white blood cells in 33 patients with retinoblastoma was detected with 3.8kb probe derived from 3' end of retinoblastoma gene cDNA. The gene abnormalities, including deletion, partial deletion and rearrangement, were found in 18 patients. Further research will be aimed at microdeletions or mutations for those patients wti...

  16. Using C-arm x-ray imaging to guide local reporter probe delivery for tracking stem cell engraftment.

    Science.gov (United States)

    Kedziorek, Dorota A; Solaiyappan, Meiyappan; Walczak, Piotr; Ehtiati, Tina; Fu, Yingli; Bulte, Jeff W M; Shea, Steven M; Brost, Alexander; Wacker, Frank K; Kraitchman, Dara L

    2013-01-01

    Poor cell survival and difficulties with visualization of cell delivery are major problems with current cell transplantation methods. To protect cells from early destruction, microencapsulation methods have been developed. The addition of a contrast agent to the microcapsule also could enable tracking by MR, ultrasound, and X-ray imaging. However, determining the cell viability within the microcapsule still remains an issue. Reporter gene imaging provides a way to determine cell viability, but delivery of the reporter probe by systemic injection may be hindered in ischemic diseases. In the present study, mesenchymal stem cells (MSCs) were transfected with triple fusion reporter gene containing red fluorescent protein, truncated thymidine kinase (SPECT/PET reporter) and firefly luciferase (bioluminescence reporter). Transfected cells were microencapsulated in either unlabeled or perfluorooctylbromide (PFOB) impregnated alginate. The addition of PFOB provided radiopacity to enable visualization of the microcapsules by X-ray imaging. Before intramuscular transplantation in rabbit thigh muscle, the microcapsules were incubated with D-luciferin, and bioluminescence imaging (BLI) was performed immediately. Twenty-four and forty-eight hours post transplantation, c-arm CT was used to target the luciferin to the X-ray-visible microcapsules for BLI cell viability assessment, rather than systemic reporter probe injections. Not only was the bioluminescent signal emission from the PFOB-encapsulated MSCs confirmed as compared to non-encapsulated, naked MSCs, but over 90% of injection sites of PFOB-encapsulated MSCs were visible on c-arm CT. The latter aided in successful targeting of the reporter probe to injection sites using conventional X-ray imaging to determine cell viability at 1-2 days post transplantation. Blind luciferin injections to the approximate location of unlabeled microcapsules resulted in successful BLI signal detection in only 18% of injections. In conclusion

  17. 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.

  18. BIOLUMINESCENCE TOMOGRAPHY: BIOMEDICAL BACKGROUND, MATHEMATICAL THEORY, AND NUMERICAL APPROXIMATION

    Institute of Scientific and Technical Information of China (English)

    Weimin Han; Ce Wang

    2008-01-01

    Over the last couple of years molecular imaging has been rapidly developed to study physiological and pathological processes in vivo at the cellular and molecular levels. Among molecular imaging modalities, optical imaging stands out for its unique advantages, especially performance and cost-effectiveness. Bioluminescence tomography (BLT) is an emerging optical imaging mode with promising biomedical advantages. In this survey paper, we explain the biomedical significance of BLT, summarize theoretical results on the analysis and numerical solution of a diffusion based BLT model, and comment on a few extensions for the study of BLT.

  19. Real-time Monitoring of Non-specific Toxicity Using a Saccharomyces cerevisiae Reporter System

    Directory of Open Access Journals (Sweden)

    Matti Karp

    2008-10-01

    Full Text Available Baker’s yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzo-flavone, rapamycin, nystatin and cycloheximide at concentrations of nM to µM. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results.

  20. Molecular insights on the evolution of the lateral and head lantern luciferases and bioluminescence colors in Mastinocerini railroad-worms (Coleoptera: Phengodidae).

    Science.gov (United States)

    Arnoldi, Frederico G C; da Silva Neto, Antonio Joaquim; Viviani, Vadim R

    2010-01-01

    Among bioluminescent beetles of Elateroidea superfamily, railroad-worms (Phengodidae) produce the widest range of colors, from green to red, using the same luciferin-luciferase system. Members of the Mastinocerini tribe display additional unique cephalic organs that emit red-shifted light, with Phrixothrix railroad-worms being the most dramatic cases with head lanterns emitting red light. Although the luciferases from the head lanterns of Phrixothrix hirtus and from the lateral lanterns of P. vivianii were previously cloned, the luciferases from both lanterns of the same species were not cloned yet. Therefore the origin and evolution of head and lateral lanterns luciferases in Phengodidae remains unknown. In the present work, we cloned by PCR the cDNA for lateral lantern luciferases of three Mastinocerini species: Phrixothrix hirtus, Brasilocerus sp(3). and Taximastioncerus sp. The results suggest that the head and lateral lanterns luciferases in Mastinocerini are coded by paralogous genes, and that the ancestral luciferase in the Phengodinae subfamily produced green bioluminescence. The evolutionary history of bioluminescence colors within Phengodinae is discussed.

  1. In vivo visualization and monitoring of viable neural stem cells using noninvasive bioluminescence imaging in the 6-hydroxydopamine-induced mouse model of Parkinson disease.

    Science.gov (United States)

    Im, Hyung-Jun; Hwang, Do Won; Lee, Han Kyu; Jang, Jaeho; Lee, Song; Youn, Hyewon; Jin, Yeona; Kim, Seung U; Kim, E Edmund; Kim, Yong Sik; Lee, Dong Soo

    2013-06-01

    Transplantation of neural stem cells (NSCs) has been proposed as a treatment for Parkinson disease (PD). The aim of this study was to monitor the viability of transplanted NSCs expressing the enhanced luciferase gene in a mouse model of PD in vivo. The PD animal model was induced by unilateral injection of 6-hydroxydopamine (6-OHDA). The behavioral test using apomorphine-induced rotation and positron emission tomography with [18F]N-(3-fluoropropyl)-2'-carbomethoxy-3'-(4-iodophenyl)nortropane ([18F]FP-CIT) were conducted. HB1.F3 cells transduced with an enhanced firefly luciferase retroviral vector (F3-effLuc cells) were transplanted into the right striatum. In vivo bioluminescence imaging was repeated for 2 weeks. Four weeks after transplantation, [18F]FP-CIT PET and the rotation test were repeated. All 6-OHDA-injected mice showed markedly decreased [18F]FP-CIT uptake in the right striatum. Transplanted F3-effLuc cells were visualized on the right side of the brain in all mice by bioluminescence imaging. The bioluminescence intensity of the transplanted F3-effLuc cells gradually decreased until it was undetectable by 10 days. The behavioral test showed that stem cell transplantation attenuated the motor symptoms of PD. No significant change was found in [18F]FP-CIT imaging after cell transplantation. We successfully established an in vivo bioluminescence imaging system for the detection of transplanted NSCs in a mouse model of PD. NSC transplantation induced behavioral improvement in PD model mice.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jae Hoon; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea); Department of Tumor Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Laboratory of Molecular Imaging and Therapy, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Kang, Joo Hyun; Jeong, Jae Min; Lee, Dong Soo [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea); Kim, Chul Woo [Department of Pathology, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Lee, Myung Chul [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea)

    2004-03-01

    Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D{sub 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 ([{sup 125}I]IVDU) and {sup 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 [{sup 125}I]IVDU and {sup 131}I, and {sup 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 {sup 125}I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [{sup 125}I]IVDU than CT-26 and CTN. NIS gene expression and {sup 125}I accumulation were found to be directly correlated (R{sup 2}=0.923), as were HSV1-tk gene expression and [{sup 125}I]IVDU accumulation (R{sup 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 {sup 131}I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [{sup 125}I]IVDU than CT-26 and CTN. Scintigraphy using {sup 131}I easily visualised CTN and

  3. A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.

    Science.gov (United States)

    Kim, Sung Bae; Ozawa, Takeaki; Umezawa, Yoshio

    2016-01-01

    We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity. PMID:27489781

  4. How synthetic biology will reconsider natural bioluminescence and its applications.

    Science.gov (United States)

    Reeve, Benjamin; Sanderson, Theo; Ellis, Tom; Freemont, Paul

    2014-01-01

    As our understanding of natural biological systems grows, so too does our ability to alter and rebuild them. Synthetic biology is the application of engineering principles to biology in order to design and construct novel biological systems for specific applications. Bioluminescent organisms offer a treasure trove of light-emitting enzymes that may have applications in many areas of bioengineering, from biosensors to lighting. A few select bioluminescent organisms have been well researched and the molecular and genetic basis of their luminescent abilities elucidated, with work underway to understand the basis of luminescence in many others. Synthetic biology will aim to package these light-emitting systems as self-contained biological modules, characterize their properties, and then optimize them for use in other chassis organisms. As this catalog of biological parts grows, synthetic biologists will be able to engineer complex biological systems with the ability to emit light. These may use luminescence for an array of disparate functions, from providing illumination to conveying information or allowing communication between organisms. PMID:25216951

  5. Bioluminescence-based imaging technique for pressure measurement in water

    Science.gov (United States)

    Watanabe, Yasunori; Tanaka, Yasufumi

    2011-07-01

    The dinoflagellate Pyrocystis lunula emits light in response to water motion. We developed a new imaging technique for measuring pressure using plankton that emits light in response to mechanical stimulation. The bioluminescence emitted by P. lunula was used to measure impact water pressure produced using weight-drop tests. The maximum mean luminescence intensity correlated with the maximum impact pressure that the cells receive when the circadian and diurnal biological rhythms are appropriately controlled. Thus, with appropriate calibration of experimentally determined parameters, the dynamic impact pressure can be estimated by measuring the cell-flash distribution. Statistical features of the evolution of flash intensity and the probability distribution during the impacting event, which are described by both biological and mechanical response parameters, are also discussed in this paper. The practical applicability of this bioluminescence imaging technique is examined through a water drop test. The maximum dynamic pressure, occurring at the impact of a water jet against a wall, was estimated from the flash intensity of the dinoflagellate.

  6. Evaluation of an improved bioluminescence assay for the detection of bacteria in soy milk.

    Science.gov (United States)

    Shinozaki, Yohei; Sato, Jun; Igarashi, Toshinori; Suzuki, Shigeya; Nishimoto, Kazunori; Harada, Yasuhiro

    2013-01-01

    Because soy milk is nutrient rich and nearly neutral in pH, it favors the growth of microbial contaminants. To ensure that soy milk meets food-safety standards, it must be pasteurized and have its sterility confirmed. ATP bioluminescence assay has become a widely accepted means of detecting food microorganisms. However, the high background bioluminescence intensity of soy milk has rendered it unsuitable for ATP analysis. Here, we tested the efficacy of an improved pre-treated bioluminescence assay on soy milk. By comparing background bioluminescence intensities obtained by the conventional and improved methods, we demonstrated that our method significantly reduces soy milk background bioluminescence. The dose-response curve of the assay was tested with serial dilutions of Bacillus sp. culture. An extremely strong log-linear relation between the bioluminescence intensity relative light units and colony formation units CFU/ml emerged for the tested strain. The detection limit of the assay was estimated as 5.2×10(3) CFU/ml from the dose-response curve and an imposed signal limit was three times the background level. The results showed that contaminated samples could be easily detected within 24 h using our improved bioluminescence assay.

  7. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    Science.gov (United States)

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments.

  8. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    Directory of Open Access Journals (Sweden)

    Jade Vacquié-Garcia

    Full Text Available How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES (Mirounga leonina have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments.

  9. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    Science.gov (United States)

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments. PMID:22952706

  10. 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.

  11. Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation.

    Science.gov (United States)

    Fortin, Pierre-Yves; Genevois, Coralie; Chapolard, Mathilde; Santalucía, Tomàs; Planas, Anna M; Couillaud, Franck

    2014-02-01

    Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response. PMID:24575340

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

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Yusuke; Okubo, Toshiyuki [University of Tokyo, Department of Radiology, Institute of Medical Science, Tokyo (Japan); Tojo, Arinobu; Sekine, Rieko; Soda, Yasushi; Kobayashi, Seiichiro; Nomura, Akiko; Izawa, Kiyoko [University of Tokyo, Division of Molecular Therapy, Advanced Clinical Research Centre, Tokyo (Japan); Kitamura, Toshio [University of Tokyo, Division of Cellular Therapy, Advanced Clinical Research Centre, Tokyo (Japan); Ohtomo, Kuni [University of Tokyo, Department of Radiology, Graduate School of Medicine, Tokyo (Japan)

    2006-05-15

    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.)

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

    International Nuclear Information System (INIS)

    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.)

  14. In Vivo Bioluminescence Imaging of Tumor Cells Using Optimized Firefly Luciferase luc2

    OpenAIRE

    2013-01-01

    The present study was aimed to establish a tumor cell line stably expressing luciferase luc2, and to develop the technique to observe primary tumor nodes and metastases using in vivo bioluminescence imaging. Materials and Methods. In this research we used pLuc2-N plasmid, lentiviral vector pLVT-1, Colo 26 cell line and BALB/c mice to generate new bioluminescent tumor model. Bioluminescence imaging in vitro и in vivo was carried out on IVIS-Spectrum system (Caliper Life Sciences, USA). Pri...

  15. Study of firefly luciferin oxidation and isomerism as possible inhibition pathways for firefly bioluminescence

    Science.gov (United States)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-01-01

    Firefly bioluminescence presents a light emitting profile with a form of a flash, due to the firefly luciferase-catalyzed formation of inhibitory products. These impair the binding of the substrate luciferin to the active site of the enzyme. However, this luciferase catalyzed pathways may not be the only ones responsible for the flash profile. The oxidation and isomerisation of the substrate luciferin lead to the formation of compounds that are also known inhibitors of firefly bioluminescence. So, the objective of this Letter was to analyze if these reactions could be capable of interfering with the bioluminescence reaction.

  16. DEVELOPMENT OF A DUAL MODALITY TOMOGRAPHIC IMAGING SYSTEM FOR BIOLUMINESCENCE AND PET

    Energy Technology Data Exchange (ETDEWEB)

    CHATZIIOANNOU, ARION

    2011-12-21

    The goal of this proposal was to develop a new hybrid imaging modality capable to simultaneously image optical bioluminescence signals, as well as radionuclide emissions from the annihilation of positrons originating from molecular imaging probes in preclinical mouse models. This new technology enables the simultaneous in-vivo measurements of both emissions that could be produced from a single or a combination of two different biomarkers. It also facilitates establishing the physical limitations of bioluminescence imaging, its tomographic and spectral image reconstruction potential and the quantification of bioluminescence signals.

  17. High resolution in vitro bioluminescence imaging using a multimodal optical system

    Science.gov (United States)

    Altabella, L.; Gigliotti, C. R.; Perani, L.; Crippa, M. P.; Boschi, F.; Spinelli, A. E.

    2016-01-01

    Bioluminescence in vitro studies are usually performed with dedicated microscopes. In this work, we developed a novel image recovery algorithm and a multimodal system prototype to perform bioluminescence microscopy. We performed a feasibility study using GEANT4 Monte Carlo (MC) simulation of bioluminescent cells acquired at low SNR frames and processed using a Super Resolution Regularization Algorithm (SRRA). The method was also tested using in vitro cell acquisition. The results obtained with MC simulations showed an improvement in the spatial resolution from 90 μ m to 10 μ m and from 110 μ m to 13 μ m for in vitro imaging of mesothelioma cells.

  18. Influence of culture conditions on mycelial growth and bioluminescence of Gerronema viridilucens.

    Science.gov (United States)

    Mendes, Luiz F; Bastos, Erick L; Desjardin, Dennis E; Stevani, Cassius V

    2008-05-01

    Herein we describe a procedure for measuring the total light emission of the naturally bioluminescent tropical fungus Gerronema viridilucens and the optimization of culture conditions using multivariate factorial anova. Cultures growing on an agar surface in 35 mm Petri dishes at 90% humidity show optimal bioluminescence emission at 25 degrees C in the presence of 1.0% sugar cane molasses, 0.10% yeast extract and pH 6.0 (nonbuffered). Temperature and pH are the most important factors for both mycelial growth and bioluminescence. PMID:18355288

  19. A multi-phase level set framework for source reconstruction in bioluminescence tomography

    International Nuclear Information System (INIS)

    We propose a novel multi-phase level set algorithm for solving the inverse problem of bioluminescence tomography. The distribution of unknown interior source is considered as piecewise constant and represented by using multiple level set functions. The localization of interior bioluminescence source is implemented by tracing the evolution of level set function. An alternate search scheme is incorporated to ensure the global optimal of reconstruction. Both numerical and physical experiments are performed to evaluate the developed level set reconstruction method. Reconstruction results show that the proposed method can stably resolve the interior source of bioluminescence tomography.

  20. High resolution in vitro bioluminescence imaging using a multimodal optical system

    International Nuclear Information System (INIS)

    Bioluminescence in vitro studies are usually performed with dedicated microscopes. In this work, we developed a novel image recovery algorithm and a multimodal system prototype to perform bioluminescence microscopy. We performed a feasibility study using GEANT4 Monte Carlo (MC) simulation of bioluminescent cells acquired at low SNR frames and processed using a Super Resolution Regularization Algorithm (SRRA). The method was also tested using in vitro cell acquisition. The results obtained with MC simulations showed an improvement in the spatial resolution from 90 μm to 10 μm and from 110 μm to 13 μm for in vitro imaging of mesothelioma cells

  1. A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

    Science.gov (United States)

    Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

    2004-03-01

    Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

  2. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

    Science.gov (United States)

    Chu, Jun; Oh, Younghee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J; Laviv, Tal; Welf, Erik S; Dean, Kevin M; Zhang, Feijie; Kim, Benjamin B; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A; Davidson, Michael W; Kay, Mark A; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S; Ng, Ho-Leung; Lin, Michael Z

    2016-07-01

    Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

  3. Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae

    OpenAIRE

    Nieto Gutiérrez, Joaquín José; Vargas, C.; Ventosa Ucero, Antonio; Arvanitis, Nikilaos; Tegos, Georgios; Perysinakis, Angelos; Drainas, Constantin

    1995-01-01

    The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pH...

  4. Bioluminescence Imaging Cells Labeled with Membrane-Anchored Form of Gaussia Luciferase%膜锚定Gaussia萤光素酶的细胞标记及生物荧光成像

    Institute of Scientific and Technical Information of China (English)

    樊炜; 贾帅争; 王怡; 阎少多; 高博; 彭剑淳; 詹林盛

    2011-01-01

    目的:制备表达膜锚定Gaussia萤光素酶(extGluc)报告基因的慢病毒,用于标记细胞.方法:将报告基因extGluc克隆至慢病毒载体pCCsin.PPT.SFFV.IRES.eGFP.Wpre (VeGFP)中,以聚乙烯亚胺(PEI)介导,将慢病毒包装所需4种质粒(pVeGFP-extGLuc、pMDL、pRey、pVSVG),转染293FT细胞,72 h后收集病毒上清进行浓缩,感染293FT细胞,并用流式细胞仪检测病毒滴度,生物荧光成像和化学发光分析extGluc的表达;之后,用收集的慢病毒感染人单核细胞白血病细胞株U937.结果:对经PCR筛选出的阳性克隆所含质粒进行酶切鉴定,表明extGlu报告基因插入载体中;重组慢病毒包装成功且病毒滴度为5×106 TU/mL;用包装的病毒颗粒感染293FT、细胞,生物荧光成像和化学发光证实extGluc的膜定位,且酶活性与细胞数目呈线性相关;病毒颗粒能够感染悬浮细胞U937.结论:包装了extGluc标记的重组慢病毒,可用于标记细胞,为体内监测细胞迁移、聚集和变化提供了一种方法.%Objective: To produce lentivirus expressing reporter gene membrane-anchored form of Gaussia lu-ciferase (extGluc) used for bioluminescence imaging cells. Methods: Reporter gene extGluc was cloned into pCCsin. PPT.SFFV.IRES.eGFP.Wpre(VeGFP). pVeGFP-extGLuc, pMDL, pRev and pVSVG, which were required for packaging, were cotransfected into 293FT cells mediated by polyethylenimine branched. Lentivirus was collected at 72 h post-transfection, concentrated and then was used to infect 293FT cells. Viral titer was determined by flow cytome-try. Luciferase activity was detected by bioluminescence imaging and chemiluminescence. At last, human monocytic leukemia cell line U937 were infected by viral supernatant. Results: PCR and enzyme digestion results indicated that reporter gene extGLuc was successfully cloned into VeGFP. The lentivirus was packaged successfully. The lentivirus titer was 5xlO6 TU/mL. After infecting 293FT with virus particles, ext

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

    Energy Technology Data Exchange (ETDEWEB)

    Jason Alan Gruenhagen

    2003-12-12

    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. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} 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{sup +} 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 functionalized Cd

  6. Bacterial bioluminescence response to long-term exposure to reverse osmosis treated effluents from dye industries.

    Science.gov (United States)

    Ravindran, J; Manikandan, B; Shirodkar, P V; Francis, K X; Mani Murali, R; Vethamony, P

    2014-10-01

    The bacterial bioluminescence assay is one of the novel means for toxicity detection. The bioluminescence response of 2 marine bioluminescent bacteria was tested upon their long-term exposure to 9 different reverse osmosis (RO) rejects with varying chemical composition sampled from various dye industries. Bioluminescent bacteria were cultured in the RO reject samples, at different concentrations, and their growth rate and luminescence was measured for 24 h. The RO reject samples caused sublethal effects upon exposure and retarded the growth of bacteria, confirming their toxic nature. Further, continuation of the exposure showed that the initial luminescence, though reduced, recovered and increased beyond the control cultures irrespective of cell density, and finally decreased once again. The present study emphasizes the need of evolving a long-term exposure assay and shows that the method followed in this study is suitable to evaluate the toxicants that exert delayed toxicity, using lower concentrations of toxicants as well as coloured samples.

  7. 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

  8. Submersible Data (Dive Trackpoints) 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 Johnson Sea Link II during sixteen dives of the "Bioluminescence 2009" expedition sponsored by the National Oceanic and...

  9. 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...

  10. 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...

  11. 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...

  12. Bioluminescence: A Potentially Convergent Signature of Life in Future Exploration of Europa's Subsurface Ocean

    Science.gov (United States)

    Flores Martinez, C. L.

    2014-02-01

    This presentation deals with theoretical and evolutionary aspects pertaining to the nature and degree of biological complexity that is expectable among putative organisms on Europa. Bioluminescence is suggested as a new type of biosignature.

  13. 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...

  14. An objective method to assess bioluminescent properties of selected bacterial strains

    Directory of Open Access Journals (Sweden)

    Bożena Danyluk

    2007-12-01

    Full Text Available Emission of light as a result of biochemical activities of some living bacteria Vibrio fischeri (in the past known as Photobacterium phosphoreum makes it possible to monitor environmental changes in ecosystems. Toxicity testing as an international standard operating procedure based on the use of this method has already been accepted. The bioluminescent test offers a rapid, simple and sensitive method to test a wide spectrum of chemical substances and environmental samples including water, wastewater, sludge extracts, etc. In this study, aimed at characterising and comparing bioluminescent properties, four different bacterial strains were cultivated in four different liquid mediums and temperature conditions. The bioluminescent intensity of bacterial suspensions was measured using a laboratory BioOrbit 1253 luminometer during bacteria culture. Based on obtained results and mathematical calculations of RLU (relative luminescent units values strain Photobacterium phosphoreum + NCBE medium were indicated as the variant demonstrating proper bioluminescence intensity and characteristics most suitable for further applications.

  15. Bioluminescent luciferase-modified magnetic nanoparticles as potential imaging agents for mammalian spermatozoa detection and tracking

    Science.gov (United States)

    Background: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-Q...

  16. 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.

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

    International Nuclear Information System (INIS)

    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)

  18. Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins.

    Science.gov (United States)

    Natashin, Pavel V; Markova, Svetlana V; Lee, John; Vysotski, Eugene S; Liu, Zhi-Jie

    2014-03-01

    Ca(2+) -regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca(2+) discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

  19. 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

    2016-01-01

    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. PMID:27220405

  20. Effect of the bile-acid sequestrant colestipol on postprandial serum bile-acid concentration: evaluation by bioluminescent enzymic analysis.

    Science.gov (United States)

    Rossi, S S; Wayne, M L; Smith, R B; Wright, C E; Andreadis, N A; Hofmann, A F

    1989-02-01

    Chronic ingestion of bile-acid sequestrants has been shown to decrease the serum cholesterol concentration and coronary events in hypercholesterolaemic patients. To develop improved sequestrants, a rapid, convenient method for testing the bile-acid binding efficacy of sequestrants is needed. Serum bile-acid concentrations could be used to detect bile-acid binding by an administered sequestrant, since the serum bile-acid concentration is determined largely by the rate of intestinal absorption in healthy individuals. To test this, serum bile-acid concentrations were measured at frequent intervals over 24 h in five otherwise healthy hypercholesterolaemic subjects during the ingestion of three standard meals, with or without the addition of 5 g colestipol granules administered 30 min before each meal. Total serum bile-acid concentration was measured with a previously reported bioluminescent enzymic assay, that uses a 3 alpha-hydroxysteroid dehydrogenase, an oxido-reductase, and a bacterial luciferase co-immobilized on to Sepharose beads. Bile acids in 1 ml of serum were isolated by solid-phase extraction chromatography with reversed-phase C18 cartridges. Colestipol lowered the postprandial elevation of serum bile acids by one half, with a subsequent decrease in the cumulative area under the curve. The data suggest that measurement of serum bile-acid concentrations by bioluminescence is a rapid, simple way to document the efficacy of bile-acid sequestrants.

  1. 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.

  2. Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin.

    Science.gov (United States)

    Hattori, Mitsuru; Kawamura, Genki; Kojima, Ryosuke; Kamiya, Mako; Urano, Yasuteru; Ozawa, Takeaki

    2016-06-21

    Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multipinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multilayered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues. PMID:27216493

  3. Foraging in the Darkness of the Southern Ocean: Influence of Bioluminescence on a Deep Diving Predator

    OpenAIRE

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES’s main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do s...

  4. Roles of biogenic amines in regulating bioluminescence in the Australian glowworm Arachnocampa flava.

    Science.gov (United States)

    Rigby, Lisa M; Merritt, David J

    2011-10-01

    The glowworm Arachnocampa flava is a carnivorous fly larva (Diptera) that uses light to attract prey into its web. The light organ is derived from cells of the Malpighian tubules, representing a bioluminescence system that is unique to the genus. Bioluminescence is modulated through the night although light levels change quite slowly compared with the flashing of the better-known fireflies (Coleoptera). The existing model for the neural regulation of bioluminescence in Arachnocampa, based on use of anaesthetics and ligations, is that bioluminescence is actively repressed during the non-glowing phase and the repression is partially released during the bioluminescence phase. The effect of the anaesthetic, carbon dioxide, on the isolated light organ from the present study indicates that the repression is at least partially mediated at the light organ itself rather than less directly through the central nervous system. Blocking of neural signals from the central nervous system through ligation leads to uncontrolled release of bioluminescence but light is emitted at relatively low levels compared with under anaesthesia. Candidate biogenic amines were introduced by several methods: feeding prey items injected with test solution, injecting the whole larva, injecting a ligated section containing the light organ or bathing the isolated light organ in test solution. Using these methods, dopamine, serotonin and tyramine do not affect bioluminescence output. Exposure to elevated levels of octopamine via feeding, injection or bathing of the isolated light organ indicates that it is involved in the regulation of repression. Administration of the octopamine antagonists phentolamine or mianserin results in very high bioluminescence output levels, similar to the effect of anaesthetics, but only mianserin acts directly on the light organ.

  5. Automatic Segmentation Framework of Building Anatomical Mouse Model for Bioluminescence Tomography

    OpenAIRE

    Abdullah Alali

    2013-01-01

    Bioluminescence tomography is known as a highly ill-posed inverse problem. To improve the reconstruction performance by introducing anatomical structures as a priori knowledge, an automatic segmentation framework has been proposed in this paper to extract the mouse whole-body organs and tissues, which enables to build up a heterogeneous mouse model for reconstruction of bioluminescence tomography. Finally, an in vivo mouse experiment has been conducted to evaluate this framework by using an X...

  6. A new bathyphotometer for bioluminescence measurements on the Armorican continental shelf (northeastern Atlantic)

    OpenAIRE

    Geistdoerfer, P; Vincendeau, Ma

    1999-01-01

    A new bathyphotometer has been developed by the Oceanographic Laboratory of the French Naval Academy which has performed a series of in situ bioluminescence: measurements since 1994. It is used as an additional probe to a Seabird (R) CTD. It measures planktonic bioluminescence. It is associated with a SeaTech (R) fluorometer. The Armorican shelf has particularly been investigated since July 1994. Results obtained in January and April 1995 off Brittany (France), at the entrance to the Bay of D...

  7. Foraging in the Darkness of the Southern Ocean: Influence of Bioluminescence on a Deep Diving Predator

    OpenAIRE

    Jade Vacquié-Garcia; François Royer; Anne-Cécile Dragon; Morgane Viviant; Frédéric Bailleul; Christophe Guinet

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do s...

  8. Cellular regulation of dinoflagellate bioluminescence : characterizing mechanosensitive ion channels in the signaling pathway

    OpenAIRE

    Jin, Kelly

    2012-01-01

    Dinoflagellate bioluminescence represents a dramatic response to mechanical stress found in nature. The cellular mechanisms that govern this pathway, however, are not completely understood. The objective of this thesis is to build and expand from previous studies to explore the mechanosensitive properties of dinoflagellate bioluminescence. Chapter I tests the hypothesis that the signaling pathway involves a stretch-activated component. Chapter I uses two separate, measurable types of biolumin...

  9. Evaluation of the Lumac kit for the detection of bacteriuria by bioluminescence.

    OpenAIRE

    Mackett, D; Kessock-Philip, S; Bascomb, S; Easmon, C S

    1982-01-01

    Four hundred and twenty-two urine samples were screened for significant bacteriuria using bioluminescence and microscopy of uncentrifuged urine. A smaller number of false-negatives were seen with bioluminescence (10%) than with microscopy (40%) while both techniques gave a similar number of false-positives (18%). The kit required a large amount of manual preparation, largely pipetting. With this and the short shelf-life of the reconstituted reagents, it is not suitable for small numbers of ur...

  10. Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin.

    Science.gov (United States)

    Hattori, Mitsuru; Kawamura, Genki; Kojima, Ryosuke; Kamiya, Mako; Urano, Yasuteru; Ozawa, Takeaki

    2016-06-21

    Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multipinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multilayered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues.

  11. Planktonic bioluminescence measurements in the frontal zone of Almeria-Oran (Mediterranean Sea)

    OpenAIRE

    Cussatlegras, As; Geistdoerfer, P.; Prieur, L.

    2001-01-01

    Plankton bioluminescence measurements were made in the Almeria-Oran frontal zone during December 1997 and January 1998. Vertical profiles of bioluminescence, chlorophyll fluorescence, temperature and salinity were obtained using a bathyphotometer associated with a CTD (conductivity-temperature-depth) probe on a rosette. The first leg of the cruise was a regular sampling along a cross section of the area. The second leg consisted of a repetitive sampling of twelve stations at each one of the e...

  12. Inflammatory bowel disease gene discovery. CRADA final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-09-09

    The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.

  13. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    Science.gov (United States)

    Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

    2001-03-01

    The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

  14. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

    Science.gov (United States)

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-06-14

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

  15. Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda.

    Science.gov (United States)

    Marek, Paul E; Moore, Wendy

    2015-05-19

    The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group's most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal's cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life.

  16. ATP bioluminescence rapid detection of total viable count in soy sauce.

    Science.gov (United States)

    Yan, Shou-Lei; Miao, Su-Na; Deng, Shao-Ya; Zou, Min-Juan; Zhong, Fo-Sheng; Huang, Wen-Biao; Pan, Si-Yi; Wang, Qing-Zhang

    2012-01-01

    The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10(2) -3 × 10(4) CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB-941) and 96-well plates and could analyse 50-100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce.

  17. The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi.

    Science.gov (United States)

    Weitz, H J; Ballard, A L; Campbell, C D; Killham, K

    2001-08-21

    The effects of temperature, light and pH on mycelial growth and luminescence of four naturally bioluminescent fungi were investigated. Cultures of Armillaria mellea, Mycena citricolor, Omphalotus olearius and Panellus stipticus were grown at 5 degrees C, 15 degrees C, 22 degrees C and 30 degrees C, under 24 h light, 12 h light/12 h dark and 24 h dark, and at a pH ranging from 3.5 to 7 in three separate experiments. Temperature and pH had a significant effect on mycelial growth and bioluminescence, however light did not. Bioluminescence and mycelial growth were optimum at 22 degrees C and pH 3-3.5, the exception being M. citricolor for which bioluminescence and growth were optimum at pH 5-6 and pH 4, respectively. With the exception of M. citricolor, bioluminescence and mycelial growth were greater under 24 h darkness. An understanding of the effect of culture conditions on mycelial growth and luminescence is necessary for the future application of bioluminescent fungi as biosensors.

  18. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

    Science.gov (United States)

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-01-01

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models. PMID:27297211

  19. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging

    Science.gov (United States)

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-01-01

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models. PMID:27297211

  20. 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.

  1. The plant mitochondrial mat-r gene/nad1 gene complex. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Wolstenholme, D.R.

    1994-06-01

    The authors have completed sequencing the segments (totalling 19 kb, both complementary strands) of the maize mtDNA molecule that encode the entire NADH dehydrogenase subunit (nadl) gene. They have identified nucleotides in mature transcripts of the nadl gene that are edited and have generated clones of cDNAs of entire mature (fully spliced) nadl transcripts. They have examined the relative rates of splicing in transcripts of the four nadl gene group II introns and begun examining nadl intron cDNAs to determine the extent and distribution of RNA edits in introns, in order to evaluate the possibility that intron excision and exon splicing might be editing independent.

  2. Conserved expression profiles of circadian clock-related genes in two Lemna species showing long-day and short-day photoperiodic flowering responses.

    Science.gov (United States)

    Miwa, Kumiko; Serikawa, Masayuki; Suzuki, Sayaka; Kondo, Takao; Oyama, Tokitaka

    2006-05-01

    The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light-dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution. PMID:16524874

  3. Quorum Sensing Influences Vibrio harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence

    OpenAIRE

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

    2008-01-01

    Background 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. Methodology/Principal Findings The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sensi...

  4. Quorum Sensing Influences Vibrio harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence

    OpenAIRE

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

    2008-01-01

    BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sen...

  5. Bioluminescence Imaging Reveals Dynamics of Beta Cell Loss in the Non-Obese Diabetic (NOD) Mouse Model

    OpenAIRE

    John Virostko; Armandla Radhika; Greg Poffenberger; Dula, Adrienne N.; Moore, Daniel J.; Alvin C Powers

    2013-01-01

    We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the ...

  6. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    Science.gov (United States)

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A

    2013-10-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  7. Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria

    OpenAIRE

    Pinel Corinne; Ropers Delphine; Ranquet Caroline; de Jong Hidde; Geiselmann Johannes

    2010-01-01

    Abstract Background Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data. Results We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate t...

  8. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

    Science.gov (United States)

    Choi, Yoori; Hwang, Do Won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  9. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development

    Science.gov (United States)

    Choi, Yoori; Hwang, Do won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  10. Noninvasive bioluminescence imaging of the dynamics of sanguinarine induced apoptosis via activation of reactive oxygen species

    Science.gov (United States)

    Dai, Yunpeng; Shi, Yaru; Zeng, Qi; Wang, Fu

    2016-01-01

    Most chemotherapeutic drugs exert their anti-tumor effects primarily by triggering a final pathway leading to apoptosis. Noninvasive imaging of apoptotic events in preclinical models would greatly facilitate the development of apoptosis-inducing compounds and evaluation of their therapeutic efficacy. Here we employed a cyclic firefly luciferase (cFluc) reporter to screen potential pro-apoptotic compounds from a number of natural agents. We demonstrated that sanguinarine (SANG) could induce apoptosis in a dose- and time-dependent manner in UM-SCC-22B head and neck cancer cells. Moreover, SANG-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and activation of c-Jun-N-terminal kinase (JNK) and nuclear factor-kappaB (NF-κB) signal pathways. After intravenous administration with SANG in 22B-cFluc xenograft models, a dramatic increase of luminescence signal can be detected as early as 48 h post-treatment, as revealed by longitudinal bioluminescence imaging in vivo. Remarkable apoptotic cells reflected from ex vivo TUNEL staining confirmed the imaging results. Importantly, SANG treatment caused distinct tumor growth retardation in mice compared with the vehicle-treated group. Taken together, our results showed that SANG is a candidate anti-tumor drug and noninvasive imaging of apoptosis using cFluc reporter could provide a valuable tool for drug development and therapeutic efficacy evaluation. PMID:26968950

  11. Noninvasive bioluminescence imaging of the dynamics of sanguinarine induced apoptosis via activation of reactive oxygen species.

    Science.gov (United States)

    Wang, Yan; Zhang, Beilei; Liu, Wei; Dai, Yunpeng; Shi, Yaru; Zeng, Qi; Wang, Fu

    2016-04-19

    Most chemotherapeutic drugs exert their anti-tumor effects primarily by triggering a final pathway leading to apoptosis. Noninvasive imaging of apoptotic events in preclinical models would greatly facilitate the development of apoptosis-inducing compounds and evaluation of their therapeutic efficacy. Here we employed a cyclic firefly luciferase (cFluc) reporter to screen potential pro-apoptotic compounds from a number of natural agents. We demonstrated that sanguinarine (SANG) could induce apoptosis in a dose- and time-dependent manner in UM-SCC-22B head and neck cancer cells. Moreover, SANG-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and activation of c-Jun-N-terminal kinase (JNK) and nuclear factor-kappaB (NF-κB) signal pathways. After intravenous administration with SANG in 22B-cFluc xenograft models, a dramatic increase of luminescence signal can be detected as early as 48 h post-treatment, as revealed by longitudinal bioluminescence imaging in vivo. Remarkable apoptotic cells reflected from ex vivo TUNEL staining confirmed the imaging results. Importantly, SANG treatment caused distinct tumor growth retardation in mice compared with the vehicle-treated group. Taken together, our results showed that SANG is a candidate anti-tumor drug and noninvasive imaging of apoptosis using cFluc reporter could provide a valuable tool for drug development and therapeutic efficacy evaluation.

  12. Analysis of Gene Targeting & Nonhomologous End-joining. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Haber, J. E.

    2002-11-30

    Overall, we identified a number of new proteins that participate in nonhomologous end-joining and also in telomere addition to the ends of broken chromosomes. We showed that NHEJ is severely reduced in cells expressing both yeast mating-type genes and then went on to identify the NEJ1 gene that was under this control. We showed the epistasis relations among a set of mutations that impair telomere addition and we showed that there are in fact two pathways to repair broken chromosomes in the absence of telomerase. We characterized the DNA damage checkpoint pathway in response to a single broken chromosome and characterized especially the adaptation of cells arrested by an unrepaired DSB. We demonstrated that the DNA damage response is nuclear-limited. We showed adaptation defects for Tid1and Srs2 proteins and showed that Srs2 was also recovery-defective, even when DNA was repaired.

  13. Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria

    Directory of Open Access Journals (Sweden)

    Pinel Corinne

    2010-04-01

    Full Text Available Abstract Background Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data. Results We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in Escherichia coli. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems. Conclusions The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

  14. A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.

    Science.gov (United States)

    Bacconi, Marta; Haag, Andreas F; Torre, Antonina; Castagnetti, Andrea; Chiarot, Emiliano; Delany, Isabel; Bensi, Giuliano

    2016-04-01

    In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens. PMID:26685857

  15. Efficient transient expression of the $\\beta$-glucuronidase reporter gene in garlic (Allium sativum L.)

    OpenAIRE

    Ferrer, Esther; Linares, Concha; González, Juan,

    2000-01-01

    International audience A biolistic particle delivery system was used to introduce DNA containing a $\\beta$-glucuronidase (gus) reporter gene under the control of the CaMV35S promoter in three different garlic (Allium sativum L.) tissues: embryogenic calli, leaves and basal plate discs. Expression of the reporter gene was assayed histochemically and fluorimetrically when the tissues were bombarded with 1 $\\mu$m diameter gold particles coated with DNA, at a distance of 3 cm from the stopping...

  16. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  17. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    OpenAIRE

    West, David B.; Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Stephen M Griffey; Engelhard, Eric K.; Rapp, Jared; LI, BOWEN; de Jong, Pieter J.; Lloyd, K. C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of...

  18. Bioluminescence flow visualization in the ocean: an initial strategy based on laboratory experiments

    Science.gov (United States)

    Rohr, Jim; Hyman, Mark; Fallon, Stewart; Latz, Michael I.

    2002-11-01

    Observations of flow-stimulated bioluminescence have been recorded for centuries throughout the world's oceans. The present study explores, within a laboratory context, the use of naturally occurring bioluminescence as a strategy towards visualizing oceanic flow fields. The response of luminescent plankton to quantifiable levels of flow agitation was investigated in fully developed pipe flow. With two different pipe flow apparatus and freshly collected mixed plankton samples obtained over a year at two separate locations, several repeatable response patterns were identified. Threshold levels for bioluminescence stimulation occurred in laminar flow with wall shear stress levels generally between 1 and 2 dyn cm -2 (0.1-0.2 N m -2), equivalent to energy dissipation per unit mass values of 10 2-10 3 cm 2 s -3 (10 -2-10 -1 m 2 s -3). In an attempt to account for different concentrations and assemblages of mixed plankton, mean bioluminescence levels were normalized by an index of the corresponding flow-stimulated bioluminescence potential. This procedure generally accounted for variability between turbulent flow experiments, but was not effective for laminar flow. In turbulent flow, mean bioluminescence levels increased approximately linearly with wall shear stress. The magnitude of the flash response of individual cells, however, remained nearly constant throughout high laminar and turbulent flow, even as the energetic length scales of the turbulence became less than the size of the organisms of interest. Threshold flow stimuli levels determined in the laboratory were compared with oceanic measurements taken from the literature and with numerical simulations of ship wakes, one of the few highly turbulent flows to be well studied. Several oceanic flow fields are proposed as candidates for bioluminescence flow visualization.

  19. Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes

    OpenAIRE

    Miller, William G; Brandl, Maria T; Quiñones, Beatriz; Lindow, Steven E.

    2001-01-01

    A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was obse...

  20. 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.

  1. 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

    RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011...... 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...... embryonic kidney HEK293t cells was used because they are easy to transfect and generally show high expression of transfected genes. Various types of fish including albino trouts and transparent fish were used as animal models to get better visualization of reporter gene expression. High variability...

  2. 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

  3. Monitoring transplanted stem cells in rat Achilles tendon by in vivo bioluminescent imaging%活体生物发光成像追踪大鼠跟腱内移植干细胞**☆○

    Institute of Scientific and Technical Information of China (English)

    黄德清; Gary Balian

    2013-01-01

    Achil es tendon of rats. METHODS: Adipose tissue derived stem cel s isolated from the abdominal cavity of Sprague-Dawley rat were transduced with an adenovirus containing the luciferase reporter gene (3×1010/L), to observe the influence of transfection on the adipose tissue derived stem cel s. Subsequently, the transfected cel s were implanted into Achil es tendon defects in rats. The in vivo bioluminescent imaging system was used at days 1, 4, 7 and 14 fol owing transplantation to assess the luciferase expression. The cryosections of repaired Achil es tendon of rats were observed under fluorescence microscope at day 28 postoperatively. RESULTS AND CONCLUSION: No influence on the morphology and proliferation of adipose tissue derived stem cel s was observed after transducing in vitro (P > 0.05). On the repaired Achil es tendon, the luciferase gene expression detected with in vivo bioluminescent imaging system at days 1, 4, 7 and 14 was respectively (1.22±0.43)×106, (1.81±0.76)×106, (1.88±0.69)×106 and (0.89±0.26)×105 counts/s (n=6). Abundant adipose tissue derived stem cel s with luciferase expression were also seen in tendon cryosections of this side under fluorescence microscope at day 28. The luciferase gene expression was not detected in the control side. Experimental findings demonstrate that the in vivo bioluminescent imaging system can successful y monitor the fluorogene modified adipose tissue derived stem cel s that are implanted into the rat Achil es tendon, and adipose tissue derived stem cel s are a potential seed cel s in tendon tissue engineering.

  4. 活体生物发光成像追踪大鼠跟腱内移植干细胞**☆○%Monitoring transplanted stem cells in rat Achilles tendon by in vivo bioluminescent imaging

    Institute of Scientific and Technical Information of China (English)

    黄德清; Gary Balian

    2013-01-01

      BACKGROUND: The mechanisms for the homing, migration, proliferation and differentiation of transplanted adipose tissue derived stem cel s remain unclear. The in vivo bioluminescent imaging system is a newly developed technique for directly detecting the biological behaviors of transplanted cel s in vivo. OBJECTIVE: To demonstrate the feasibility of using in vivo bioluminescent imaging system to monitor the genetical y modified adipose tissue derived stem cel s transplanted in Achil es tendon of rats. METHODS: Adipose tissue derived stem cel s isolated from the abdominal cavity of Sprague-Dawley rat were transduced with an adenovirus containing the luciferase reporter gene (3×1010/L), to observe the influence of transfection on the adipose tissue derived stem cel s. Subsequently, the transfected cel s were implanted into Achil es tendon defects in rats. The in vivo bioluminescent imaging system was used at days 1, 4, 7 and 14 fol owing transplantation to assess the luciferase expression. The cryosections of repaired Achil es tendon of rats were observed under fluorescence microscope at day 28 postoperatively. RESULTS AND CONCLUSION: No influence on the morphology and proliferation of adipose tissue derived stem cel s was observed after transducing in vitro (P > 0.05). On the repaired Achil es tendon, the luciferase gene expression detected with in vivo bioluminescent imaging system at days 1, 4, 7 and 14 was respectively (1.22±0.43)×106, (1.81±0.76)×106, (1.88±0.69)×106 and (0.89±0.26)×105 counts/s (n=6). Abundant adipose tissue derived stem cel s with luciferase expression were also seen in tendon cryosections of this side under fluorescence microscope at day 28. The luciferase gene expression was not detected in the control side. Experimental findings demonstrate that the in vivo bioluminescent imaging system can successful y monitor the fluorogene modified adipose tissue derived stem cel s that are implanted into the rat Achil es tendon, and

  5. 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.

  6. Molecular basis for the blue bioluminescence of the Australian glow-worm Arachnocampa richardsae (Diptera: Keroplatidae).

    Science.gov (United States)

    Trowell, Stephen C; Dacres, Helen; Dumancic, Mira M; Leitch, Virginia; Rickards, Rodney W

    2016-09-16

    Bioluminescence is the emission of visible light by living organisms. Here we describe the isolation and characterisation of a cDNA encoding a MW ≈ 59,000 Da luciferase from the Australian glow-worm, Arachnocampa richardsae. The enzyme is a member of the acyl-CoA ligase superfamily and produces blue light on addition of D-luciferin. These results are contrary to earlier reports (Lee, J., Photochem Photobiol 24, 279-285 (1976), Viviani, V. R., Hastings, J. W. & Wilson, T., Photochem Photobiol 75, 22-27 (2002)), which suggested glow-worm luciferase has MW ≈ 36,000 Da and is unreactive with beetle luciferin. There are more than 2000 species of firefly, which all produce emissions from D-luciferin in the green to red regions of the electromagnetic spectrum. Although blue-emitting luciferases are known from marine organisms, they belong to different structural families and use a different substrate. The observation of blue emission from a D-luciferin-using enzyme is therefore unprecedented. PMID:27457804

  7. Molecular basis for the blue bioluminescence of the Australian glow-worm Arachnocampa richardsae (Diptera: Keroplatidae).

    Science.gov (United States)

    Trowell, Stephen C; Dacres, Helen; Dumancic, Mira M; Leitch, Virginia; Rickards, Rodney W

    2016-09-16

    Bioluminescence is the emission of visible light by living organisms. Here we describe the isolation and characterisation of a cDNA encoding a MW ≈ 59,000 Da luciferase from the Australian glow-worm, Arachnocampa richardsae. The enzyme is a member of the acyl-CoA ligase superfamily and produces blue light on addition of D-luciferin. These results are contrary to earlier reports (Lee, J., Photochem Photobiol 24, 279-285 (1976), Viviani, V. R., Hastings, J. W. & Wilson, T., Photochem Photobiol 75, 22-27 (2002)), which suggested glow-worm luciferase has MW ≈ 36,000 Da and is unreactive with beetle luciferin. There are more than 2000 species of firefly, which all produce emissions from D-luciferin in the green to red regions of the electromagnetic spectrum. Although blue-emitting luciferases are known from marine organisms, they belong to different structural families and use a different substrate. The observation of blue emission from a D-luciferin-using enzyme is therefore unprecedented.

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

    International Nuclear Information System (INIS)

    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

  9. Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice.

    Science.gov (United States)

    Li, Xiao-Feng; Deng, Yong-Qiang; Zhao, Hui; Ye, Qing; Wang, Hong-Jiang; Li, Shi-Hua; Zhu, Shun-Ya; Shi, Pei-Yong; Qin, E-De; Zhang, Bo; Qin, Cheng-Feng

    2013-05-01

    Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R(2) = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.

  10. High Rates of Species Accumulation in Animals with Bioluminescent Courtship Displays.

    Science.gov (United States)

    Ellis, Emily A; Oakley, Todd H

    2016-07-25

    One of the great mysteries of evolutionary biology is why closely related lineages accumulate species at different rates. Theory predicts that populations undergoing strong sexual selection will more quickly differentiate because of increased potential for genetic isolation [1-6]. Whether or not these population genetic processes translate to more species at macroevolutionary scales remains contentious [7]. Here we show that lineages with bioluminescent courtship, almost certainly a sexually selected trait, have more species and faster rates of species accumulation than their non-luminous relatives. In each of ten distantly related animal lineages from insects, crustaceans, annelid worms, and fishes, we find more species in lineages with bioluminescent courtship compared to their sister groups. Furthermore, we find under a Yule model that lineages with bioluminescent courtship displays have significantly higher rates of species accumulation compared to a larger clade that includes them plus non-luminous relatives. In contrast, we do not find more species or higher rates in lineages that use bioluminescence for defense, a function presumably not under sexual selection. These results document an association between the origin of bioluminescent courtship and increased accumulation of species, supporting theory predicting sexual selection increases rates of speciation at macroevolutionary scales to influence global patterns of biodiversity. PMID:27345160

  11. Development of bioluminescent bioreporters for in vitro and in vivo tracking of Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Yanwen Sun

    Full Text Available Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux(PtolC and Lux(PcysZK. These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux(PtolC and monitor intracellular survival (Lux(PtolC and Lux(PcysZK in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.

  12. Spectrally resolved bioluminescence tomography with the third-order simplified spherical harmonics approximation

    International Nuclear Information System (INIS)

    Bioluminescence imaging has been extensively applied to in vivo small animal imaging. Quantitative three-dimensional bioluminescent source information obtained by using bioluminescence tomography can directly and much more accurately reflect biological changes as opposed to planar bioluminescence imaging. Preliminary simulated and experimental reconstruction results demonstrate the feasibility and promise of bioluminescence tomography. However, the use of multiple approximations, particularly the diffusion approximation theory, affects the quality of in vivo small animal-based image reconstructions. In the development of new reconstruction algorithms, high-order approximation models of the radiative transfer equation and spectrally resolved data introduce new challenges to the reconstruction algorithm and speed. In this paper, an SP3-based (the third-order simplified spherical harmonics approximation) spectrally resolved reconstruction algorithm is proposed. The simple linear relationship between the unknown source distribution and the spectrally resolved data is established in this algorithm. A parallel version of this algorithm is realized, making BLT reconstruction feasible for the whole body of small animals especially for fine spatial domain discretization. In simulation validations, the proposed algorithm shows improved reconstruction quality compared with diffusion approximation-based methods when high absorption, superficial sources and detection modes are considered. In addition, comparisons between fine and coarse mesh-based BLT reconstructions show the effects of numerical errors in reconstruction image quality. Finally, BLT reconstructions using in vivo mouse experiments further demonstrate the potential and effectiveness of the SP3-based reconstruction algorithm.

  13. First report of the blaVIM gene in environmental isolates of Buttiauxella sp.

    Science.gov (United States)

    Pitondo-Silva, André; Martins, Vinicius Vicente; Stehling, Eliana Guedes

    2015-04-01

    Several works have demonstrated the presence of metallo-β-lactamases (MBLs) in clinical bacteria. However, in environmental isolates, few works have reported on these enzymes. In this study, we report for the first time two environmental isolates of Buttiauxella sp. recovered from chrysanthemum plantations in Brazil containing blaVIM gene and producing MBLs.

  14. A multithread based new sparse matrix method in bioluminescence tomography

    Science.gov (United States)

    Zhang, Bo; Tian, Jie; Liu, Dan; Sun, Li; Yang, Xin; Han, Dong

    2010-03-01

    Among many molecular imaging modalities, bioluminescence tomography (BLT) stands out as an effective approach for in vivo imaging because of its noninvasive molecular and cellular level detection ability, high sensitivity and low cost in comparison with other imaging technologies. However, there exists the case that large scale problem with large number of points and elements in the structure of mesh standing for the small animal or phantom. And the large scale problem's system matrix generated by the diffuse approximation (DA) model using finite element method (FEM) is large. So there wouldn't be enough random access memory (RAM) for the program and the related inverse problem couldn't be solved. Considering the sparse property of the BLT system matrix, we've developed a new sparse matrix (ZSM) to overcome the problem. And the related algorithms have all been speeded up by multi-thread technologies. Then the inverse problem is solved by Tikhonov regularization method in adaptive finite element (AFE) framework. Finally, the performance of this method is tested on a heterogeneous phantom and the boundary data is obtained through Monte Carlo simulation. During the process of solving the forward model, the ZSM can save more processing time and memory space than the usual way, such as those not using sparse matrix and those using Triples or Cross Linked sparse matrix. Numerical experiments have shown when more CPU cores are used, the processing speed is increased. By incorporating ZSM, BLT can be applied to large scale problems with large system matrix.

  15. Bioluminescence to reveal structure and interaction of coastal planktonic communities

    Science.gov (United States)

    Moline, Mark A.; Blackwell, Shelley M.; Case, James F.; Haddock, Steven H. D.; Herren, Christen M.; Orrico, Cristina M.; Terrill, Eric

    2009-02-01

    Ecosystem function will in large part be determined by functional groups present in biological communities. The simplest distinction with respect to functional groups of an ecosystem is the differentiation between primary and secondary producers. A challenge thus far has been to examine these groups simultaneously with sufficient temporal and spatial resolution for observations to be relevant to the scales of change in coastal oceans. This study takes advantage of general differences in the bioluminescence flash kinetics between planktonic dinoflagellates and zooplankton to measure relative abundances of the two groups within the same-time space volume. This novel approach for distinguishing these general classifications using a single sensor is validated using fluorescence data and exclusion experiments. The approach is then applied to data collected from an autonomous underwater vehicle surveying >500 km in Monterey Bay and San Luis Obispo Bay, CA during the summers of 2002-2004. The approach also reveals that identifying trophic interaction between the two planktonic communities may also be possible.

  16. Report of a chimeric origin of transposable elements in a bovine-coding gene.

    Science.gov (United States)

    Almeida, L M; Amaral, M E J; Silva, I T; Silva, W A; Riggs, P K; Carareto, C M

    2008-02-01

    Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is approximately 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.

  17. 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. PMID:27468205

  18. Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

    Science.gov (United States)

    Magalhães, Carla M; Esteves da Silva, Joaquim C G; Pinto da Silva, Luís

    2016-08-01

    Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment. PMID:27129132

  19. Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

    Science.gov (United States)

    Magalhães, Carla M; Esteves da Silva, Joaquim C G; Pinto da Silva, Luís

    2016-08-01

    Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.

  20. 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.

  1. Design and implementation of an optical simulation environment for bioluminescent tomography studies

    Institute of Scientific and Technical Information of China (English)

    LI Hui; TIAN Jie; LUO Jie; L(U) Yujie; CONG Wenxiang; WANG Ge

    2007-01-01

    As a challenging task for bioluminescent tomography simulation, a virtual optical environment is needed to solve the forward problem accurately, that is, to achieve a high precision for bioluminescent signal synthesis on the external body surface of a small animal. The molecular optical simulation environment named MOSE is implemented using the C + + programming language and the OpenGL techniques, including a user-friendly interface with interactive tools facilitating users' operations. The accuracy of the virtual optical environment is verified by error analysis of mesh simplification and comparison between MOSE results and experimental data. This virtual optical environment is accurate, flexible and efficient to simulate the photon propagation in complicated tissues, which has a great potential to become a software platform for bioluminescent tomography studies and other molecular imaging applications.

  2. Quantification of adipose transfer viability using a novel, bioluminescent murine model.

    Science.gov (United States)

    Gassman, Andrew A; Kao, Kenneth K; Bradley, James P; Lee, Justine C

    2016-07-01

    Fat grafting has highly variable long-term results. Research efforts to improve the reliability of fat grafting are limited by inefficient methods for evaluation of fat engraftment. In this work, we describe a novel animal model for the quantitative evaluation of fat grafting using in vivo bioluminescence of adipocytes from luciferase-expressing mice. Subcutaneous adipose tissue from GFP and luciferase-expressing FVB mice were obtained. The samples were homogenized, decanted, and injected into the dorsal skin folds of wild-type FVB mice. Viability of the transferred tissue was examined over a 28-day time period with quantitative bioluminescence after luciferin injection. All animals demonstrated viable adipose transfer with bioluminescence detectable on days 0, 1, 7, 14, and 28. This animal model may be used for noninvasive, longitudinal studies for quantification of the fat engraftment process. PMID:27017232

  3. 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. PMID:25979598

  4. The First Bioluminescence Tomography System for Simultaneous Acquisition of Multiview and Multispectral Data

    Directory of Open Access Journals (Sweden)

    Ge Wang

    2006-01-01

    Full Text Available We describe the system design of the first bioluminescence tomography (BLT system for parallel acquisition of multiple bioluminescent views around a mouse in a number of spectral channels simultaneously. The primary component of this BLT system is a novel mirror module and a unique mouse holder. The mirror module consists of a mounting plate and four mirrors with stages. These mirror stages are right triangular blocks symmetrically arranged and attached to the mounting plate such that the hypotenuse surfaces of the triangular blocks all make 45∘ to the plate surface. The cylindrical/polygonal mouse holder has semitransparent rainbow bands on its side surface for the acquisition of spectrally resolved data. Numerical studies and experiments are performed to demonstrate the feasibility of this system. It is shown that bioluminescent signals collected using our system can produce a similar BLT reconstruction quality while reducing the data acquisition time, as compared to the sequential data acquisition mode.

  5. Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

    Directory of Open Access Journals (Sweden)

    Catherine E Winbanks

    Full Text Available Recombinant adeno-associated viral vectors (rAAV vectors are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal, human placental alkaline phosphatase (hPLAP, and green fluorescent protein (GFP as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

  6. In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice

    International Nuclear Information System (INIS)

    Transgenic mice expressing the luciferase (luc) gene under the control of the heme oxygenase-1 promoter (Ho1) were used to measure the induction of heme oxygenase in response to known toxicants. Transgenic Ho1-luc expression was visualized in vivo using a low-light imaging system (IVIS). Ho1-luc activation was compared to Ho1-luc expression, HO1 protein levels, standard markers of toxicity, and histology. Male and female Ho1-luc transgenic mice were exposed to acute doses of cadmium chloride (CdCl2, 3.7 mg/kg), doxorubicin (15 mg/kg), and thioacetamide (300 mg/kg). These agents induced the expression of Ho1-luc in the liver and other tissues to varying degrees. The greatest increase in Ho1-luc activity was observed in the liver in response to CdCl2; intermediate responses were observed for doxorubicin and thioacetamide. Induction of the Ho1-luc transgene by these agents was similar to endogenous protein levels of heme oxygenase as assessed by Western blotting, and generally correlated with plasma levels of circulating enzymes reflecting hepatic or general tissue damage. Histopathology confirmed the toxic effects of CdCl2 on liver and kidney; doxorubicin on kidney, liver, and intestine; and thioacetamide on the liver. Tissue damage was much more pronounced than the luciferase expression following thioacetamide treatment when compared with tissue damage and bioluminescence of the other toxicants. Nevertheless, the induction of Ho1-luc expression following exposure to these agents suggests that the Ho1-luc transgenic mouse may prove useful as a model for in vivo screening of compounds that induce luciferase expression as a marker of toxicity

  7. 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

    . 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...... results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi....

  8. 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.

  9. Bioluminescent bioreporter integrated circuit devices and methods for detecting estrogen

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

    2006-08-15

    Bioelectronic devices for the detection of estrogen include a collection of eukaryotic cells which harbor a recombinant lux gene from a high temperature microorganism wherein the gene is operably linked with a heterologous promoter gene. A detectable light-emitting lux gene product is expressed in the presence of the estrogen and detected by the device.

  10. Formulation of photon diffusion from spherical bioluminescent sources in an infinite homogeneous medium

    Directory of Open Access Journals (Sweden)

    Wang Lihong V

    2004-05-01

    Full Text Available Abstract Background The bioluminescent enzyme firefly luciferase (Luc or variants of green fluorescent protein (GFP in transformed cells can be effectively used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies can be evaluated by using bioluminescent imaging. In bioluminescent imaging, light propagates in highly scattering tissue, and the diffusion approximation is sufficiently accurate to predict the imaging signal around the biological tissue. The numerical solutions to the diffusion equation take large amounts of computational time, and the studies for its analytic solutions have attracted more attention in biomedical engineering applications. Methods Biological tissue is a turbid medium that both scatters and absorbs photons. An accurate model for the propagation of photons through tissue can be adopted from transport theory, and its diffusion approximation is applied to predict the imaging signal around the biological tissue. The solution to the diffusion equation is formulated by the convolution between its Green's function and source term. The formulation of photon diffusion from spherical bioluminescent sources in an infinite homogeneous medium can be obtained to accelerate the forward simulation of bioluminescent phenomena. Results The closed form solutions have been derived for the time-dependent diffusion equation and the steady-state diffusion equation with solid and hollow spherical sources in a homogeneous medium, respectively. Meanwhile, the relationship between solutions with a solid sphere source and ones with a surface sphere source is obtained. Conclusion We have formulated solutions for the diffusion equation with solid and hollow spherical sources in an infinite homogeneous medium. These solutions have been verified by Monte Carlo simulation for use in biomedical optical imaging studies. The closed form solution is highly accurate and more

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

    International Nuclear Information System (INIS)

    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

  12. Gene transcription and electromagnetic fields. Final progress report

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, A.S.

    1992-12-31

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

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

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of); Kim, Yeon Soo [Schoole of Medicine, Inje University, Seoul (Korea, Republic of)

    2004-07-01

    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.

  14. 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.

  15. Effects of salinity, pH and temperature on the re-establishment of bioluminescence and copper or SDS toxicity in the marine dinoflagellate Pyrocystis lunula using bioluminescence as an endpoint

    International Nuclear Information System (INIS)

    The bioluminescence assay is not sensitive to small changes in pH, temperature or salinity. - Pyrocystis lunula is a unicellular, marine, photoautotrophic, bioluminescent dinoflagellate. This organism is used in the Lumitox[reg] bioassay with inhibition of bioluminescence re-establishment as the endpoint. Experiments determined if acute changes in pH, salinity, or temperature had an effect on the organisms' ability to re-establish bioluminescence, or on the bioassay's potential to detect sodium dodecyl sulfate (SDS) and copper toxicity. The re-establishment of bioluminescence itself was not very sensitive to changes in pH within the pH 6-10 range, though reducing pH from 8 to levels below 6 decreased this capacity. Increasing the pH had little effect on Cu or SDS toxicity, but decreasing the pH below 7 virtually eliminated the toxicity of either compound in the bioassay. Lowering the salinity from 33 to 27%o or less resulted in a substantial decrease in re-establishment of bioluminescence, while increasing the salinity to 43 or 48 %o resulted in a small decline. Salinity had little influence on the bioassay's quantification of Cu toxicity, while the data showed a weak negative relationship between SDS toxicity and salinity. Re-establishment of bioluminescence showed a direct dependence on temperature, but only at 10 deg. C did temperature have an obvious effect on the toxicity of Cu in this bioassay

  16. A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression.

    OpenAIRE

    Feng, B.; Friedlin, E; Marzluf, G A

    1994-01-01

    Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused ...

  17. Optimization of reporter gene assay: several factors influencing detection of promoter activity

    Institute of Scientific and Technical Information of China (English)

    XUE Li-xiang; WENG Mo; ZHANG Zong-yu; TONG Tan-jun

    2007-01-01

    Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.Methods Human embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination,such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA.Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

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

    Directory of Open Access Journals (Sweden)

    Gabriela Kuncová

    2016-06-01

    Full Text Available Living cells of the lux-based bioluminescent bioreporter Pseudomonas putida TVA8 were encapsulated in a silica hydrogel attached to the distal wider end of a tapered quartz fiber. Bioluminescence of immobilized cells was induced with toluene at high (26.5 mg/L and low (5.3 mg/L concentrations. Initial bioluminescence maxima were achieved after >12 h. One week after immobilization, a biofilm-like layer of cells had formed on the surface of the silica gel. This resulted in shorter response times and more intensive bioluminescence maxima that appeared as rapidly as 2 h after toluene induction. Considerable second bioluminescence maxima were observed after inductions with 26.5 mg toluene/L. The second and third week after immobilization the biosensor repetitively and semiquantitatively detected toluene in buffered medium. Due to silica gel dissolution and biofilm detachment, the bioluminescent signal was decreasing 20–32 days after immobilization and completely extinguished after 32 days. The reproducible formation of a surface cell layer on the wider end of the tapered optical fiber can be translated to various whole cell bioluminescent biosensor devices and may serve as a platform for in-situ sensors.

  19. 3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

    Institute of Scientific and Technical Information of China (English)

    Jie Jing; Men Jing; Wang Chun-mei; Gao Xue-jun; Li Qing-zhang

    2012-01-01

    To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3' UTR was predicted by Target Scan 5.1 software, 3' UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3' UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3' UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.

  20. 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 ER-

  1. Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay

    NARCIS (Netherlands)

    Freitas, de J.; Cano, P.; Craig-Veit, C.; Goodson, M.L.; Furlow, J.D.; Murk, A.J.

    2011-01-01

    A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensi

  2. Multiple insulin degrading enzyme variants alter in vitro reporter gene expression.

    Directory of Open Access Journals (Sweden)

    Olivia Belbin

    Full Text Available The insulin degrading enzyme (IDE variant, v311 (rs6583817, is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ, decreased risk for Alzheimer's disease (AD and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957, for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2 successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04 but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5' to the promoter (1.18 fold-change, p = 0.006 and 3' to the reporter gene (1.29 fold change, p = 0.003, an effect contributed to by four variants (v10, v315, v154 and v311. Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.

  3. Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy

    Directory of Open Access Journals (Sweden)

    Gonzalez-Aparicio Manuela

    2009-01-01

    Full Text Available Abstract Background Bioluminescent imaging (BLI is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies. Results A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1 retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US, BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12. Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival. Conclusion We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

  4. 生物发光成像的特点及应用%Bioluminescence imaging characteristics and application

    Institute of Scientific and Technical Information of China (English)

    杨丽平; 赵敬湘; 裴雪涛

    2009-01-01

    生物发光成像(BLI)是通过荧光素酶基因标记细胞或DNA,在ATP及氧气存在条件下,催化荧光素的氧化反应而发光,从而能够直接监控活体内的细胞活动和基因行为.该文通过比较BLI与MRI,PET、放射成像的异同,以及BLI在肿瘤、干细胞和免疫细胞运输、细胞凋亡等方面的应用,为更好地推广BLI的应用提供依据.%Bioluminescence imaging (BLI) by luciferase gene marked cells or DNA, in the presence of ATP and oxygen, catalytic oxidation reaction of fluorescein luminescence. So that it can directly monitor in vivo cell activity and gene behavior. In this paper, by comparing the BLI and MRI, PET, radiography of the similarities and differences, as well as about their cancer, stem cells and immune cells transportation, apoptosis and other aspects of the application, in order to better provide the basis for promoting the application of BLI.

  5. Detection of light and vibration modulates bioluminescence intensity in the glowworm, Arachnocampa flava.

    Science.gov (United States)

    Mills, Rebecca; Popple, Julie-Anne; Veidt, Martin; Merritt, David John

    2016-04-01

    Glowworms are larval fungus gnats that emit light from a specialised abdominal light organ. The light attracts small arthropod prey to their web-like silk snares. Larvae glow throughout the night and can modulate their bioluminescence in response to sensory input. To better understand light output regulation and its ecological significance, we examined the larvae's reaction to light exposure, vibration and sound. Exposure to a 5-min light pulse in the laboratory causes larvae to exponentially decrease their light output over 5-10 min until they completely switch off. They gradually return to pre-exposure levels but do not show a rebound. Larvae are most sensitive to ultraviolet light, then blue, green and red. Vibration of the larval snares results in a several-fold increase in bioluminescence over 20-30 s, followed by an exponential return to pre-exposure levels over 15-30 min. Under some conditions, larvae can respond to vibration by initiating bioluminescence when they are not glowing; however, the response is reduced compared to when they are glowing. We propose that inhibitory and excitatory mechanisms combine to modulate bioluminescence intensity by regulating biochemical reactions or gating the access of air to the light organ. PMID:26897608

  6. Three-dimensional localization of in vivo bioluminescent source based on multispectral imaging

    Science.gov (United States)

    Feng, Jinchao; Jia, Kebin; Tian, Jie; Yan, Guorui; Zhu, Shouping

    2009-02-01

    Bioluminescence tomography (BLT) is a novel in vivo technique in small animal studies, which can reveal the molecular and cellular information at the whole-body small animal level. At present, there is an increasing interest in multispectral bioluminescence tomography, since multispectral data acquisition could improve the BLT performance significantly. In view to the ill-posedness of BLT problem, we develop an optimal permissible source region strategy to constrain the possible solution of the source by utilizing spectrum character of bioluminescent source. Then a linear system to link the measured data with the unknown light source variables is established by utilizing the optimal permissible region strategy based on adaptive finite element analysis. Furthermore, singular value decomposition analysis is used for data dimensionality reduction and improving computational efficiency in multispectral case. The reconstructed speed and stability benefit from adaptive finite element, the permissible region strategy and singular value decomposition. In the numerical simulation, the heterogeneous phantom experiment has been used to evaluate the performance of the proposed algorithm with the Monte Carlo based synthetic data. The reconstruction results demonstrate the merits and potential of our methodology for localizing bioluminescent source.

  7. Detection of light and vibration modulates bioluminescence intensity in the glowworm, Arachnocampa flava.

    Science.gov (United States)

    Mills, Rebecca; Popple, Julie-Anne; Veidt, Martin; Merritt, David John

    2016-04-01

    Glowworms are larval fungus gnats that emit light from a specialised abdominal light organ. The light attracts small arthropod prey to their web-like silk snares. Larvae glow throughout the night and can modulate their bioluminescence in response to sensory input. To better understand light output regulation and its ecological significance, we examined the larvae's reaction to light exposure, vibration and sound. Exposure to a 5-min light pulse in the laboratory causes larvae to exponentially decrease their light output over 5-10 min until they completely switch off. They gradually return to pre-exposure levels but do not show a rebound. Larvae are most sensitive to ultraviolet light, then blue, green and red. Vibration of the larval snares results in a several-fold increase in bioluminescence over 20-30 s, followed by an exponential return to pre-exposure levels over 15-30 min. Under some conditions, larvae can respond to vibration by initiating bioluminescence when they are not glowing; however, the response is reduced compared to when they are glowing. We propose that inhibitory and excitatory mechanisms combine to modulate bioluminescence intensity by regulating biochemical reactions or gating the access of air to the light organ.

  8. 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 o

  9. Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

    Science.gov (United States)

    Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

    2014-10-17

    ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

  10. A Monte-Carlo-Based Network Method for Source Positioning in Bioluminescence Tomography

    OpenAIRE

    Zhun Xu; Xiaolei Song; Xiaomeng Zhang; Jing Bai

    2007-01-01

    We present an approach based on the improved Levenberg Marquardt (LM) algorithm of backpropagation (BP) neural network to estimate the light source position in bioluminescent imaging. For solving the forward problem, the table-based random sampling algorithm (TBRS), a fast Monte Carlo simulation method ...

  11. Establishment of a bioluminescence model for microenvironmentally induced oral carcinogenesis with implications for screening bioengineered scaffolds

    Science.gov (United States)

    Suliman, Salwa; Parajuli, Himalaya; Sun, Yang; Johannessen, Anne Christine; Finne–Wistrand, Anna; McCormack, Emmet; Mustafa, Kamal

    2015-01-01

    Abstract Background Microenvironmental cues play a major role in head and neck cancer. Biodegradable scaffolds used for bone regeneration might also act as stimulative cues for head and neck cancer. The purpose of this study was to establish an experimental model for precise and noninvasive evaluation of tumorigenic potential of microenvironmental cues in head and neck cancer. Methods Bioluminescence was chosen to image tumor formation. Early neoplastic oral keratinocyte (DOK) cells were luciferase‐transduced (DOKLuc), then tested in nonobese diabetic severe combined immunodeficient IL2rγnull mice either orthotopically (tongue) or subcutaneously for their potential as “screening sensors” for diverse microenvironmental cues. Results Tumors formed after inoculation of DOKLuc were monitored easier by bioluminescence, and bioluminescence was more sensitive in detecting differences between various microenvironmental cues when compared to manual measurements. Development of tumors from DOKLuc grown on scaffolds was also successfully monitored noninvasively by bioluminescence. Conclusion The model presented here is a noninvasive and sensitive model for monitoring the impact of various microenvironmental cues on head and neck cancer in vivo. © 2015 The Authors Head & Neck Published by Wiley Periodicals, Inc. Head Neck 38: E1177–E1187, 2016 PMID:26275210

  12. Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

    Directory of Open Access Journals (Sweden)

    Andrea Osimani

    2014-10-01

    Full Text Available ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs, including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99 between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

  13. Effect of quiones and phenols on the triple-enzyme bioluminescent system with protease

    NARCIS (Netherlands)

    Kudruasheva, N.S.; Esimbekova, E.N.; Remmel, N.N.; Kratasyuk, V.A.; Visser, A.J.W.G.; Hoek, van A.

    2003-01-01

    The study addressed the effects of redox-active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox-active compounds. Trypsin activity was quantified by bioluminescent technique. Interactions of these compounds with trypsin were stu

  14. Noctiluca sp. bioluminescence in response to the mechanical stimuli of a screw propeller

    Directory of Open Access Journals (Sweden)

    Han Jing

    2015-01-01

    Full Text Available This work presents a new experiment method studying the Noctiluca sp. bioluminescence under the mechanical stimulation. It devoted to the study of the Noctiluca sp. bioluminescence triggered by the screw propeller’s mechanical stimuli in the tank. The size of the tank was 2*1*1m. The screw propeller is fixed on a shelf and the position relative to the tank was adjustable by moving the shelf. Two methods were carried out to control the running of the screw propeller. In the first scenario, the shelf was fixed in the center of the tank and the second scenario, the shelf moved from one side to the other in the tank. At the same time, the screw propeller was running with a certain velocity. The luminescent strength of Noctiluca sp. enhanced as the increase of the screw propeller’s running velocity. There were two obvious luminous areas nearby the screw propeller’s blades. The luminescent area was bigger in the second scenario. Thus, when navigational ship passing the sea area which filled with Noctiluca sp. or other luminescent halobios, it will stimulate the Noctiluca sp. or other luminescent halobios bioluminescence. The ship also can be detected using the bioluminescence.

  15. 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.

  16. Rapid detection of bacteria in green tea using a novel pretreatment method in a bioluminescence assay.

    Science.gov (United States)

    Shinozaki, Yohei; Harada, Yasuhiro

    2014-06-01

    Tea is one of the most popular beverages consumed in the world, and green tea has become a popular beverage in Western as well as Asian countries. A novel pretreatment method for a commercial bioluminescence assay to detect bacteria in green tea was developed and evaluated in this study. Pretreatment buffers with pH levels ranging from 6.0 to 9.0 were selected from MES (morpholineethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), or Tricine buffers. To evaluate the effect of pretreatment and the performance of the assay, serially diluted cultures of Enterobacter cloacae, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus were tested. The improved methods, which consisted of a pretreatment of the sample in alkaline buffer, significantly decreased the background bioluminescence intensity of green tea samples when compared with the conventional method. Pretreatment with alkaline buffers with pH levels ranging from 8.0 to 9.0 increased the bioluminescence intensities of cultures of E. cloacae and S. aureus. Strong log-linear relationships between the bioluminescence intensities and plate counts emerged for the tested strains. Furthermore, the microbial detection limit was 15 CFU in 500 ml of bottled green tea after an 8-h incubation at 35°C and an assay time of 1 h. The results showed that contaminated samples could be detected within 1 h of operation using our improved bioluminescence assay. This method could be used to test for contamination during the manufacturing process as well as for statistical sampling for quality control.

  17. Bioluminescence enhancement through an added washing protocol enabling a greater sensitivity to carbofuran toxicity.

    Science.gov (United States)

    Jia, Kun; Eltzov, Evgeni; Marks, Robert S; Ionescu, Rodica E

    2013-10-01

    The effects of carbofuran toxicity on a genetically modified bacterial strain E. coli DPD2794 were enhanced using a new bioluminescent protocol which consisted of three consecutive steps: incubation, washing and luminescence reading. Specifically, in the first step, several concentrations of carbofuran aqueous solutions were incubated with different bacterial suspensions at recorded optical densities for different lengths of time. Thereafter, the resulting bacterial/toxicant mixtures were centrifuged and the aged cellular supernatant replaced with fresh medium. In the final step, the carbofuran- induced bioluminescence to the exposed E. coli DPD2794 bacteria was shown to provide a faster and higher intensity when recorded at a higher temperature at30°C which is not usually used in the literature. It was found that the incubation time and the replacement of aged cellular medium were essential factors to distinguish different concentrations of carbofuran in the bioluminescent assays. From our results, the optimum incubation time for a "light ON" bioluminescence detection of the effect of carbofuran was 6h. Thanks to the replacement of the aged cellular medium, a group of additional peaks starting around 30min were observed and we used the corresponding areas under the curve (AUC) at different contents of carbofuran to produce the calibration curve. Based on the new protocol, a carbofuran concentration of 0.5pg/mL can be easily determined in a microtiter plate bioluminescent assay, while a non-wash protocol provides an unexplainable order of curve evolutionswhich does not allow the user to determine the concentration. PMID:23867093

  18. Bioluminescence enhancement through an added washing protocol enabling a greater sensitivity to carbofuran toxicity.

    Science.gov (United States)

    Jia, Kun; Eltzov, Evgeni; Marks, Robert S; Ionescu, Rodica E

    2013-10-01

    The effects of carbofuran toxicity on a genetically modified bacterial strain E. coli DPD2794 were enhanced using a new bioluminescent protocol which consisted of three consecutive steps: incubation, washing and luminescence reading. Specifically, in the first step, several concentrations of carbofuran aqueous solutions were incubated with different bacterial suspensions at recorded optical densities for different lengths of time. Thereafter, the resulting bacterial/toxicant mixtures were centrifuged and the aged cellular supernatant replaced with fresh medium. In the final step, the carbofuran- induced bioluminescence to the exposed E. coli DPD2794 bacteria was shown to provide a faster and higher intensity when recorded at a higher temperature at30°C which is not usually used in the literature. It was found that the incubation time and the replacement of aged cellular medium were essential factors to distinguish different concentrations of carbofuran in the bioluminescent assays. From our results, the optimum incubation time for a "light ON" bioluminescence detection of the effect of carbofuran was 6h. Thanks to the replacement of the aged cellular medium, a group of additional peaks starting around 30min were observed and we used the corresponding areas under the curve (AUC) at different contents of carbofuran to produce the calibration curve. Based on the new protocol, a carbofuran concentration of 0.5pg/mL can be easily determined in a microtiter plate bioluminescent assay, while a non-wash protocol provides an unexplainable order of curve evolutionswhich does not allow the user to determine the concentration.

  19. Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Sonja Schallenberg

    Full Text Available CD4(+CD25(+ regulatory T (Treg cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4(+CD8(+ double-positive (DP and CD4(+CD8(? single-positive stages of thymic development, as well as in postthymic CD4(+ T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3(+ Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3(+ DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4(+ T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3(+ Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3(+ Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

  20. Molecular characterization of a maize regulatory gene. Progress report, July 1989--March 1990

    Energy Technology Data Exchange (ETDEWEB)

    Wessler, S.

    1990-12-31

    This progress report contains information concerning the characterization of the Maize regulatory gene. The findings of this research program have immediate significance. Firstly, it provides support for the notion that R proteins, produced by the regulatory gene, are functionally equivalent. Secondly, the success of these experiments provides a simple transient assay for either natural or constructed R protein mutations. The relative ease of this assay coupled with overnight results are important prerequisites to the proposed experiments involving a structure-function analysis of the R protein.

  1. 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......-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription, To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast...

  2. An assessment of recently published gene expression data analyses: reporting experimental design and statistical factors

    Directory of Open Access Journals (Sweden)

    Azuaje Francisco

    2006-06-01

    Full Text Available Abstract Background The analysis of large-scale gene expression data is a fundamental approach to functional genomics and the identification of potential drug targets. Results derived from such studies cannot be trusted unless they are adequately designed and reported. The purpose of this study is to assess current practices on the reporting of experimental design and statistical analyses in gene expression-based studies. Methods We reviewed hundreds of MEDLINE-indexed papers involving gene expression data analysis, which were published between 2003 and 2005. These papers were examined on the basis of their reporting of several factors, such as sample size, statistical power and software availability. Results Among the examined papers, we concentrated on 293 papers consisting of applications and new methodologies. These papers did not report approaches to sample size and statistical power estimation. Explicit statements on data transformation and descriptions of the normalisation techniques applied prior to data analyses (e.g. classification were not reported in 57 (37.5% and 104 (68.4% of the methodology papers respectively. With regard to papers presenting biomedical-relevant applications, 41(29.1 % of these papers did not report on data normalisation and 83 (58.9% did not describe the normalisation technique applied. Clustering-based analysis, the t-test and ANOVA represent the most widely applied techniques in microarray data analysis. But remarkably, only 5 (3.5% of the application papers included statements or references to assumption about variance homogeneity for the application of the t-test and ANOVA. There is still a need to promote the reporting of software packages applied or their availability. Conclusion Recently-published gene expression data analysis studies may lack key information required for properly assessing their design quality and potential impact. There is a need for more rigorous reporting of important experimental

  3. A mouse model for studying the clearance of hepatitis B virus in vivo using a luciferase reporter.

    Directory of Open Access Journals (Sweden)

    Sheng-qiang Liang

    Full Text Available Hepatitis B virus(HBV infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc. We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.

  4. Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects of Environmental Chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Bonefeld-Jørgensen, Eva Cecilie; Larsen, John Christian

    1999-01-01

    cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV)2-luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold...... induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p′-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional...... cancer cells with an estrogen response element–luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties....

  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. Effects of salinity, pH and temperature on the re-establishment of bioluminescence and copper or SDS toxicity in the marine dinoflagellate Pyrocystis lunula using bioluminescence as an endpoint

    Science.gov (United States)

    Craig, J.M.; Klerks, P.L.; Heimann, K.; Waits, J.L.

    2003-01-01

    Pyrocystis lunula is a unicellular, marine, photoautotrophic, bioluminescent dinoflagellate. This organism is used in the Lumitox ?? bioassay with inhibition of bioluminescence re-establishment as the endpoint. Experiments determined if acute changes in pH, salinity, or temperature had an effect on the organisms' ability to re-establish bioluminescence, or on the bioassay's potential to detect sodium dodecyl sulfate (SDS) and copper toxicity. The re-establishment of bioluminescence itself was not very sensitive to changes in pH within the pH 6-10 range, though reducing pH from 8 to levels below 6 decreased this capacity. Increasing the pH had little effect on Cu or SDS toxicity, but decreasing the pH below 7 virtually eliminated the toxicity of either compound in the bioassay. Lowering the salinity from 33 to 27??? or less resulted in a substantial decrease in re-establishment of bioluminescence, while increasing the salinity to 43 or 48 ??? resulted in a small decline. Salinity had little influence on the bioassay's quantification of Cu toxicity, while the data showed a weak negative relationship between SDS toxicity and salinity. Re-establishment of bioluminescence showed a direct dependence on temperature, but only at 10??C did temperature have an obvious effect on the toxicity of Cu in this bioassay. ?? 2003 Elsevier Science Ltd. All rights reserved.

  7. Bioluminescent bioreporter integrated circuit devices and methods for detecting ammonia

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L [Knoxville, TN; Paulus, Michael J [Knoxville, TN; Sayler, Gary S [Blaine, TN; Applegate, Bruce M [West Lafayette, IN; Ripp, Steven A [Knoxville, TN

    2007-04-24

    Monolithic bioelectronic devices for the detection of ammonia includes a microorganism that metabolizes ammonia and which harbors a lux gene fused with a heterologous promoter gene stably incorporated into the chromosome of the microorganism and an Optical Application Specific Integrated Circuit (OASIC). The microorganism is generally a bacterium.

  8. Effects of Binary Mixtures of Inducers (Toluene Analogs) and of Metals on Bioluminescence Induction of a Recombinant Bioreporter Strain

    OpenAIRE

    In Chul Kong

    2014-01-01

    This paper investigated the effects of binary mixtures of bioluminescence inducers (toluene, xylene isomers, m-toluate) and of metals (Cu, Cd, As(III), As(V), and Cr) on bioluminescence activity of recombinant (Pm-lux) strain KG1206. Different responses and sensitivities were observed depending on the types and concentrations of mixtures of inducers or metals. In the case of inducer mixtures, antagonistic and synergistic modes of action were observed, whereas metal mixtures showed all three m...

  9. A Dual-Color Far-Red to Near-Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging**

    OpenAIRE

    Jathoul, A. P.; Grounds, H.; Anderson, J.; Pule, M. A.

    2014-01-01

    Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase. However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual color, far-red to near infrared (nIR) emitting analogue of beetle luciferin, which akin to natural luciferin e...

  10. Novel mechanism of bioluminescence: oxidative decarboxylation of a moiety adjacent to the light emitter of Fridericia luciferin.

    Science.gov (United States)

    Dubinnyi, Maxim A; Kaskova, Zinaida M; Rodionova, Natalja S; Baranov, Mikhail S; Gorokhovatsky, Andrey Yu; Kotlobay, Alexey; Solntsev, Kyril M; Tsarkova, Aleksandra S; Petushkov, Valentin N; Yampolsky, Ilia V

    2015-06-01

    A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter. PMID:25913753

  11. Alteration of bioluminescence in Amphipholis squamata (Ophiuroidea: Echinodermata) by heavy metals contamination: a field study

    OpenAIRE

    Deheyn, D; Jangoux, M.; Warnau, M.

    2000-01-01

    The ophiuroid Amphipholis squamata (Echinodermata) is a bioluminescent species whose light production varies with physico-chemical parameters of the medium. Individuals collected in the bay of Portman along a gradient of heavy metal contamination show different patterns of light production: the ones from the highest contaminated area showing a bioluminescence weaker and slower than those from the lowest contaminated area. Individuals that were transferred for 3 days from the lowest to the hig...

  12. Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.

    Science.gov (United States)

    Marui, Junichiro; Yoshimi, Akira; Hagiwara, Daisuke; Fujii-Watanabe, Yoshimi; Oda, Ken; Koike, Hideaki; Tamano, Koichi; Ishii, Tomoko; Sano, Motoaki; Machida, Masayuki; Abe, Keietsu

    2010-08-01

    Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

  13. 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 TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B 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 NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-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...

  14. 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...

  15. Exploitation of Herpesviral Transactivation Allows Quantitative Reporter Gene-Based Assessment of Virus Entry and Neutralization

    OpenAIRE

    Henrike Reinhard; Vu Thuy Khanh Le; Mats Ohlin; Hartmut Hengel; Mirko Trilling

    2011-01-01

    Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expressi...

  16. Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation

    OpenAIRE

    Catherine E Winbanks; Claudia Beyer; Hongwei Qian; Paul Gregorevic

    2012-01-01

    Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipul...

  17. Assessing the effect of EPO on tumor oxygenation and radioresponsiveness via in vivo bioluminescence imaging

    International Nuclear Information System (INIS)

    Evaluating tumor kill by volume measurement lacks sensitivity while in vivo-in vitro and histological assays are unsuitable for serial measurements. In vivo bioluminescence imaging (BI) nondestructively measures the number of metabolically active cells containing luciferase (LUC) over time. In this paper, the effect of erythropoietin (EPO) on tumor oxygenation and radioresponsivenessis is studied using BI and conventional methods. Murine adenocarcinoma cells, transfected with the LUC gene, were placed in the flank of BALB/C mice. EPO 1 u/g or saline was injected sc tiw for two weeks, starting the day of transplant. Mice then underwent irradiation (XRT) or pO2 measurement with an optical probe. In BI, mice were injected with luciferin and total photon flux (TPF) measured with a CCD camera. In vitro, cells were plated, irradiated and incubated at 37 deg C. Initial hematocrit was 47% (n=119) vs. 61% in EPO-treated mice (n=23, p2 (6.4 vs. 4.7 mm Hg, p=0.04) than controls. For 1-3x7 Gy, TPF was stable for 2 days after the start of XRT, then fell precipitously. Two weeks post XRT, TPF was 10-5 the initial value and a nidus of LUC activity persisted for months in most tumors. Tumor volume decreased only 1-2 orders of magnitude. For 3x7 Gy, tumor regrew in 1/11 EPO-TM and controls (p=NS.) For 1x7 Gy, tumors regrew in 4/6 EPO-TM and 2/4 controls (p=NS). TPF did not increase with tumor regrowth. Recurrent tumors exhibited lower median pO2 (2.1 mm Hg, p=.003) and higher hypoxic fraction than controls. A clonogenic assay yielded D10 = 3.7 Gy with all colonies expressing LUC. The TPF of 0-Gy treated wells rose significantly over incubation, while that of wells treated to 10 Gy was unchanged. Though EPO improved tumor oxygenation, no effect on XRT-mediated cell kill was seen. BI measured tumor killing in vivo over a broad dynamic range. The results suggest that cell killing in vivo is a multistep process, amplified by humoral factors

  18. In vivo fluorescence imaging of the reticuloendothelial system using quantum dots in combination with bioluminescent tumour monitoring

    International Nuclear Information System (INIS)

    We characterised in vivo fluorescence imaging (FLI) of the reticuloendothelial system using quantum dots (QD) and investigated its use in combination with in vivo bioluminescence imaging (BLI). In vivo FLI was performed in five mice repeatedly after the intravenous administration of QD without conjugation to targeting ligands. Ex vivo FLI of the excised organs was performed 24 h after QD injection in three mice. Seven days after intravenous inoculation of luciferase-expressing model cells of a haematological malignancy, mice were injected with the QD or saline (n = 5 each), and combined BLI/FLI was performed repeatedly. Additional five mice inoculated with the tumour cells were examined by in vivo BLI/FLI, and the structures harbouring bioluminescent foci were determined by ex vivo BLI. The utility of combining FLI with bioluminescent tumour monitoring was evaluated. In vivo FLI after QD injection allowed long-term, repeated observation of the reticuloendothelial system in individual mice, although fluorescence intensity and image contrast gradually decreased over time. Ex vivo FLI verified selective accumulation in reticuloendothelial structures. The administration of QD did not affect whole-body bioluminescent signal intensities during longitudinal tumour monitoring. In vivo BLI/FLI, accompanied by fusion of both images, improved the accuracy and confidence level of the localisation of the bioluminescent foci. In vivo FLI using QD provides an overview of the reticuloendothelial system in living mice. In combination with bioluminescent tumour monitoring, fluorescent reticuloendothelial imaging is expected to provide valuable information for lesion localisation. (orig.)

  19. Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay.

    Science.gov (United States)

    Yoshida, Wataru; Baba, Yuji; Karube, Isao

    2016-09-20

    DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal. PMID:27541340

  20. Evaluation of monkeypox virus infection of black-tailed 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

    2014-07-01

    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. PMID:24779460

  1. Final Report [Function of the Arabidopsis TIR1 gene in auxin response

    Energy Technology Data Exchange (ETDEWEB)

    Estelle, Mark

    2000-12-18

    During this grant period substantial progress was made in the characterization of the TIR1 gene in Arabidopsis. Studies showed that the TIR1 protein is part of a protein complex that includes AtCUL1, ASK1 and RBX1. This complex, called SCF-TIR1, functions in the ubiquitin-mediated protein degradation pathway. Our work is the first report of an SCF complex in a plant system. The results of our studies are described in more detail in the report together with a publication resulting from this study.

  2. Mosaic 18q21.2 deletions including the TCF4 gene: a clinical report.

    Science.gov (United States)

    Rossi, Massimiliano; Labalme, Audrey; Cordier, Marie-Pierre; Till, Marianne; Blanchard, Gaëlle; Dubois, Remi; Guibaud, Laurent; Heissat, Sophie; Javouhey, Etienne; Lachaux, Alain; Mure, Pierre-Yves; Ville, Dorothée; Edery, Patrick; Sanlaville, Damien

    2012-12-01

    Pitt-Hopkins syndrome (PTHS) is characterized by distinctive facial dysmorphism, profound intellectual disability, and the possible occurrence of epilepsy and breathing anomalies. It is caused by haploinsufficiency of the TCF4 gene. No significant difference in clinical severity has been reported to date between PTHS patients carrying 18q21 deletions including the TCF4 gene, and those harboring TCF4 point mutations, suggesting a lack of genotype/phenotype correlation. Moreover, the size of 18q21 deletions including the TCF4 gene does not appear to have a significant effect on the phenotypic severity, suggesting that TCF4 haploinsufficiency is the most important prognostic factor in 18q deletions. We describe two unrelated patients presenting with clinical features reminiscent of PTHS and carrying mosaic interstitial 18q21 deletions characterized by array comparative genomic hybridization. One of the patients presented the lowest level of mosaic 18q21 deletion reported to date (5-10%). Our report and a review of the literature show that the mosaic status does not appear to have a significant effect on the clinical severity of 18q21 deletions, which are associated with a poor neurological outcome, whereas a mosaic TCF4 point mutation can result in a significantly milder phenotype. Malformations of internal organs are currently considered to be rare in PTHS. The patients described here had visceral anomalies, suggesting that a full morphological assessment, including heart and abdominal ultrasound scans, should be performed systematically in PTHS patients. PMID:23165966

  3. Biological sensor for sucrose availability: relative sensitivities of various reporter genes.

    Science.gov (United States)

    Miller, W G; Brandl, M T; Quiñones, B; Lindow, S E

    2001-03-01

    A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 micro

  4. First report of a de novo germline mutation in the MLH1 gene

    Institute of Scientific and Technical Information of China (English)

    Rein P Stulp; Yvonne J Vos; Bart Mol; Arend Karrenbeld; Monique de Raad; Huub JC van der Mijle; Rolf H Sijmons

    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 majority of these HNPCC-associated mutations have been proven,or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient's parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  5. 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.

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

    International Nuclear Information System (INIS)

    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

  7. 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,...

  8. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    Science.gov (United States)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  9. Improved light extraction in the bioluminescent lantern of a Photuris firefly (Lampyridae)

    Science.gov (United States)

    Bay, Annick; Cloetens, Peter; Suhonen, Heikki; Vigneron, Jean Pol

    2013-01-01

    A common problem of light sources emitting from an homogeneous high-refractive index medium into air is the loss of photons by total internal reflection. Bioluminescent organisms, as well as artificial devices, have to face this problem. It is expected that life, with its mechanisms for evolution, would have selected appropriate optical structures to get around this problem, at least partially. The morphology of the lantern of a specific firefly in the genus Photuris has been examined. The optical properties of the different parts of this lantern have been modeled, in order to determine their positive or adverse effect with regard to the global light extraction. We conclude that the most efficient pieces of the lantern structure are the misfit of the external scales (which produce abrupt roughness in air) and the lowering of the refractive index at the level of the cluster of photocytes, where the bioluminescent production takes place.

  10. 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.

  11. Biocidal effects of silver and zinc oxide nanoparticles on the bioluminescent bacteria

    Science.gov (United States)

    Taran, M. V.; Starodub, N. F.; Katsev, A. M.; Guidotti, M.; Khranovskyy, V. D.; Babanin, A. A.; Melnychuk, M. D.

    2013-11-01

    The effect of silver and zinc oxide nanoparticles in combination with alginate on bioluminescent Photobacterium leiognathi Sh1 bacteria was investigated. Silver nanoparticles were found to be more toxic than zinc oxide nanoparticles on bioluminescent bacteria. The nanoparticles and their ions released results in the same effect, however, it was absent in combination with alginate. The effective inhibiting concentration (EC50) for silver nanoparticles was found about 0.3 - 0.4 μg mL-1, which was up to two times larger then for zinc oxide nanoparticles. The absence of sodium chloride in the tested media prevented the formation of colloidal particles of larger size and the effective inhibition concentrations of metal derivatives were lower than in the presence of sodium chloride.

  12. Robust red-emission spectra and yields in firefly bioluminescence against temperature changes

    Science.gov (United States)

    Mochizuki, Toshimitsu; Wang, Yu; Hiyama, Miyabi; Akiyama, Hidefumi

    2014-05-01

    We measured the quantitative spectra of firefly (Photinus pyralis) bioluminescence at various temperatures to investigate the temperature dependence of the luciferin-luciferase reaction at 15-34 °C. The quantitative spectra were decomposed very well into red (1.9 eV), orange (2.0 eV), and green (2.2 eV) Gaussian components. The intensity of the green component was the only temperature sensitive quantity that linearly decreased as the temperature increased at pH 7 and 8. We found the quantitative bioluminescence spectra to be robust below 2.0 eV against temperature and other experimental conditions. The revealed robustness of the red emissions should be useful for quantitative applications such as adenosine-5'-triphosphate detection.

  13. Improved light extraction in the bioluminescent lantern of a Photuris firefly (Lampyridae)

    CERN Document Server

    Bay, Annick; Suhonen, Heikki; Vigneron, Jean Pol

    2012-01-01

    A common problem of light sources emitting from an homogeneous high-refractive index medium into air is the loss of photons by total internal reflection. Bioluminescent organisms, as well as artificial devices, have to face this problem. It is expected that life, with its mechanisms for evolution, would have selected appropriate optical structures to get around this problem, at least partially. The morphology of the lantern of a specific firefly in the genus Photuris has been examined. The optical properties of the different parts of this lantern have been modelled, in order to determine their positive or adverse effect with regard to the global light extraction. We conclude that the most efficient pieces of the lantern structure are the misfit of the external scales (which produce abrupt roughness in air) and the lowering of the refractive index at the level of the cluster of photocytes, where the bioluminescent production takes place.

  14. 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. PMID:27424915

  15. Application of ATP-bioluminescence assay for screening chemotherapeutic agents of ovarian cancer chemotherapy

    International Nuclear Information System (INIS)

    To evaluate the feasibility of using ATP-bioluminescence assay for tumor chemosensitivity testing in vitro, authors selected the A2780 cell line as a model and established the suitable assay condition. Screening chemotherapeutic agents of ovarian cancer in vitro were preliminarily researched. Using this assay, dose-response curve was detected in cell line treated with these agents. The result showed that the coefficients of variation for assay ranged from 0.2% to 8.2%, which means high reproducibility. It can measured ATP content of as few as forty cells. The thermal stability of the luciferin-luciferase system was high enough used in industry. The predictable accuracy rate is about 90.6%. This study demonstrated ATP-bioluminescence assay is a reliable, reproducible and sensitive method. It can provide a technical base for screening sensitive chemotherapeutic agents in clinic

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

    Science.gov (United States)

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-09-01

    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 vanC₁ and 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.

  17. 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.

  18. In vivo bioluminescence tomography based on multi-view projection and 3D surface reconstruction

    Science.gov (United States)

    Zhang, Shuang; Wang, Kun; Leng, Chengcai; Deng, Kexin; Hu, Yifang; Tian, Jie

    2015-03-01

    Bioluminescence tomography (BLT) is a powerful optical molecular imaging modality, which enables non-invasive realtime in vivo imaging as well as 3D quantitative analysis in preclinical studies. In order to solve the inverse problem and reconstruct inner light sources accurately, the prior structural information is commonly necessary and obtained from computed tomography or magnetic resonance imaging. This strategy requires expensive hybrid imaging system, complicated operation protocol and possible involvement of ionizing radiation. The overall robustness highly depends on the fusion accuracy between the optical and structural information. In this study we present a pure optical bioluminescence tomographic system (POBTS) and a novel BLT method based on multi-view projection acquisition and 3D surface reconstruction. The POBTS acquired a sparse set of white light surface images and bioluminescent images of a mouse. Then the white light images were applied to an approximate surface model to generate a high quality textured 3D surface reconstruction of the mouse. After that we integrated multi-view luminescent images based on the previous reconstruction, and applied an algorithm to calibrate and quantify the surface luminescent flux in 3D.Finally, the internal bioluminescence source reconstruction was achieved with this prior information. A BALB/C mouse with breast tumor of 4T1-fLuc cells mouse model were used to evaluate the performance of the new system and technique. Compared with the conventional hybrid optical-CT approach using the same inverse reconstruction method, the reconstruction accuracy of this technique was improved. The distance error between the actual and reconstructed internal source was decreased by 0.184 mm.

  19. Autonomously Bioluminescent Mammalian Cells For Continuous And Real-Time Monitoring Of Cytotoxicity

    OpenAIRE

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

    2013-01-01

    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 fac...

  20. Quantum Yield Determination Based on Photon Number Measurement, Protocols for Firefly Bioluminescence Reactions.

    Science.gov (United States)

    Niwa, Kazuki

    2016-01-01

    Quantum yield (QY), which is defined as the probability of photon production by a single bio/chemiluminescence reaction, is an important factor to characterize luminescence light intensity emitted diffusively from the reaction solution mixture. Here, methods to measure number of photons to determine QY according to the techniques of national radiometry standards are described. As an example, experiments using firefly bioluminescence reactions are introduced. PMID:27424895