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Sample records for bioluminescence based reporter

  1. Reconstructing promoter activity from Lux bioluminescent reporters.

    PubMed

    Iqbal, Mudassar; Doherty, Neil; Page, Anna M L; Qazi, Saara N A; Ajmera, Ishan; Lund, Peter A; Kypraios, Theodore; Scott, David J; Hill, Philip J; Stekel, Dov J

    2017-09-18

    The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

  2. 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

    PubMed Central

    Berger, Frank; Paulmurugan, Ramasamy; Bhaumik, Srabani

    2014-01-01

    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 gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated. Methods 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. Results 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. Conclusions 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

  3. Fluorescent and Bioluminescent Reporter Myxoviruses

    PubMed Central

    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

  4. A Nisin Bioassay Based on Bioluminescence

    PubMed Central

    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. PMID:10427078

  5. Detection of bacteria with bioluminescent reporter bacteriophage.

    PubMed

    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.

  6. Amplitude metrics for cellular circadian bioluminescence reporters.

    PubMed

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

    2014-12-02

    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

  7. Amplitude Metrics for Cellular Circadian Bioluminescence Reporters

    PubMed Central

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

    2014-01-01

    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

  8. Smartphone-based low light detection for bioluminescence application

    USDA-ARS?s Scientific Manuscript database

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

  9. Bioluminescence.

    ERIC Educational Resources Information Center

    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)

  10. Bioluminescence.

    ERIC Educational Resources Information Center

    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)

  11. Contribution of the cytoskeleton to mechanosensitivity reported by dinoflagellate bioluminescence.

    PubMed

    Stires, J C; Latz, M I

    2017-08-03

    The cytoskeleton is crucial to cell mechanics and sensing the extracellular physical environment. The objective of this study was to examine the role of the cortical cytoskeleton in mechanosensitivity in a unicellular protist, the marine dinoflagellate Lingulodinium polyedra, using its intrinsic bioluminescence as a rapid reporter of mechanotransduction. Pharmacological treatments resolved effects due to immediate cytoskeleton disruption from those due to cytoskeletal remodeling during the light to dark phase transition. The cytoskeleton was visualized by confocal laser scanning microscopy of immunohistochemically labeled microtubules and phalloidin labeled F-actin, and mechanosensitivity assessed based on the bioluminescence response to mechanical stimulation measured during the dark phase. Latrunculin B treatment after the transition from the light to dark phase resulted in some disruption of cortical F-actin, no observed effect on the cortical microtubules, and partial inhibition of the bioluminescence response. Treatment with oryzalin, which depolarizes microtubules, completely disrupted the microtubule network and cortical F-actin, and partially inhibited bioluminescence. These results demonstrate that cells retain some mechanosensitivity despite a disrupted cytoskeleton; link mechanosensitivity to intact F-actin; show a close connection between F-actin and microtubules comprising the cortical cytoskeleton; confirm a strong contribution of the actin cytoskeleton to the translocation of scintillons, vesicles containing the luminescent chemistry; and support the role of the actin cytoskeleton in the association of scintillons with the vacuole membrane. © 2017 Wiley Periodicals, Inc.

  12. Assessment of shikonin for potential estrogenic activity by dual-luciferase reporter based bioluminescent measurements in vitro.

    PubMed

    Zhang, Yi; Li, Ping-Ping

    2011-02-01

    Shikonin, an active component of Lithospermum erythrorhizon Sieb. et Zucc., shows multiple pharmacological properties. However, the estrogenic activity of shikonin is remaining unclear. We assessed the potential estrogenic activity of shikonin with dual-luciferase reporter assay and bioluminescent measurements, by using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells. Estrogenic activity of shikonin, even at high concentration did not alter significantly compared to negative control (p > 0.05) and were significantly lower than those with E2 (p < 0.01). Concluding, shikonin demonstrates no estrogenic activity in vitro.

  13. In Vivo Mouse Bioluminescence Tomography with Radionuclide-Based Imaging Validation

    PubMed Central

    Lu, Yujie; Machado, Hidevaldo B.; Bao, Qinan; Stout, David; Herschman, Harvey

    2010-01-01

    Introduction Bioluminescence imaging, especially planar bioluminescence imaging, has been extensively applied in in vivo preclinical biological research. Bioluminescence tomography (BLT) has the potential to provide more accurate imaging information due to its 3D reconstruction compared with its planar counterpart. Methods In this work, we introduce a positron emission tomography (PET) radionuclide imaging-based strategy to validate the BLT results. X-ray computed tomography, PET, spectrally resolved bioluminescence imaging, and surgical excision were performed on a tumor xenograft mouse model expressing a bioluminescent reporter gene. Results With different spectrally resolved measured data, the BLT reconstructions were acquired based on the third-order simplified spherical harmonics (SP3) approximation and the diffusion approximation (DA). The corresponding tomographic images were obtained for validation of bioluminescence source reconstruction. Conclusion Our results show the strength of PET imaging compared with other validation methods for BLT and improved source localization accuracy based on the SP3 approximation compared with the diffusion approximation. PMID:20464517

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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method.

    PubMed

    Han, Taewon; Wren, Melody; DuBois, Kelsey; Therkorn, Jennifer; Mainelis, Gediminas

    2015-12-01

    An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol

  17. Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method

    PubMed Central

    Han, Taewon; Wren, Melody; DuBois, Kelsey; Therkorn, Jennifer; Mainelis, Gediminas

    2015-01-01

    An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol

  18. Smartphone-based low light detection for bioluminescence application

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  19. Smartphone-based low light detection for bioluminescence application.

    PubMed

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

    2017-01-09

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~10(6) CFU/mL of the bio-reporter, which corresponds to ~10(7) photons/s with 180 seconds of integration time.

  20. Smartphone-based low light detection for bioluminescence application

    PubMed Central

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

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time. PMID:28067287

  1. Rapid, sensitive bioluminescent reporter technology for napthalene exposure and biodegradation

    SciTech Connect

    King, J.M.H.; DiGrazia, P.M.; Applegate, B.; Burlage, R.; Sanseverino, J.; Dunbar, P.; Sayler, G.S. ); Larimer, F. )

    1990-08-17

    A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferase-mediated light production from P. fluorescens strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in Manufactured Gas Plant soils and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of napthalene biodegradative activity. The robust and sensitive nature of the bioluminescent reporter technology suggests that new sensing methods can be developed for on-line process monitoring and control in complex environmental matrices.

  2. Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium

    SciTech Connect

    Heitzer, A.; Malachowsky, K.; Thonnard, J.E.

    1994-05-01

    An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical guide by using strontium alginate. The biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either napthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 fuel or an aqueous leachate from a manufactured-gas plant soil, since napthalene was present in both pollutant mixtures. 43 refs., 4 figs., 1 tab.

  3. Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

    PubMed Central

    Heitzer, A; Malachowsky, K; Thonnard, J E; Bienkowski, P R; White, D C; Sayler, G S

    1994-01-01

    An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures. PMID:8017932

  4. Isolation and development of bioluminescent reporter phages for bacterial dysentery.

    PubMed

    Schofield, D A; Wray, D J; Molineux, I J

    2015-02-01

    Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

  5. BLProt: prediction of bioluminescent proteins based on support vector machine and relieff feature selection.

    PubMed

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

    2011-08-17

    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. 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. 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. The BLProt software is available at http://www.inb.uni-luebeck.de/tools-demos/bioluminescent%20protein/BLProt.

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

    PubMed Central

    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

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

    PubMed

    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.

  8. Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode.

    PubMed

    Daniel, Ramiz; Almog, Ronen; Ron, Amit; Belkin, Shimshon; Diamand, Yosi Shacahm

    2008-12-01

    Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).

  9. Evaluation of estrogenic potential of Shu-Gan-Liang-Xue Decoction by dual-luciferase reporter based bioluminescent measurements in vitro.

    PubMed

    Zhang, Yi; Li, Ping-Ping

    2009-11-12

    Shu-Gan-Liang-Xue Decoction (SGLXD), a cipher prescription of traditional Chinese medicine, has been used to ameliorate hot flushes symptom in breast cancer patients for several decades. However, estrogenic activity of SGLXD is remaining unclear. To evaluate the estrogenic potential of SGLXD and each of the component herbs; to investigate the effect of SGLXD on cell viability of MCF-7 cells. We evaluated the estrogenic potential of SGLXD and each of the component herbs with dual-luciferase reporter assay and bioluminescent measurements, by using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells. We investigated the effect of SGLXD on cell viability of MCF-7 by MTT assay. SGLXD and each of the component herbs did not demonstrate estrogenic activity compared to negative control, even at high concentration (p > 0.05). By MTT assay, different concentrations of SGLXD significantly inhibited the growth of MCF-7 cells dose-dependently (p < 0.05). SGLXD and single herbs do not manifest estrogenic activity, while SGLXD has weak inhibitory effects on MCF-7 cell viability.

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

    PubMed

    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.

  11. BLProt: prediction of bioluminescent proteins based on support vector machine and relieff feature selection

    PubMed Central

    2011-01-01

    Background Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence. Results In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated. Conclusion BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. The BLProt software is available at http://www.inb.uni-luebeck.de/tools-demos/bioluminescent%20protein/BLProt PMID:21849049

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

    SciTech Connect

    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.

  13. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  14. Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

    PubMed

    Takeuchi, Masaki; Nagaoka, Yasutaka; Yamada, Toshimichi; Takakura, Hideo; Ozawa, Takeaki

    2010-11-15

    Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.

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

    PubMed

    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.

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

    PubMed

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

    2017-09-01

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

  17. Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging.

    PubMed

    Yeh, Hsien-Wei; Karmach, Omran; Ji, Ao; Carter, David; Martins-Green, Manuela M; Ai, Hui-Wang

    2017-09-04

    Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

  18. Bioluminescence Imaging of Reporter Mice for Studies of Infection and Inflammation

    PubMed Central

    Luker, Kathryn E.; Luker, Gary D.

    2010-01-01

    In vivo bioluminescence imaging offers the opportunity to study biological processes in living animals, and the study of viral infections and host immune responses can be enhanced substantially through this imaging modality. For most studies of viral pathogenesis and effects of anti-viral therapies, investigators have used recombinant viruses engineered to express a luciferase enzyme. This strategy requires stable insertion of an imaging reporter gene into the viral genome, which is not feasible for many RNA viruses, and provides data on the viral component of pathogenesis but not the host. Genetically-engineered mice with luciferase reporters for specific viral or host genes provide opportunities to overcome these limitations and expand applications of bioluminescence imaging in viral infection and therapy. We review several different types of reporter mice for bioluminescence imaging, including animals that permit in vivo detection of viral replication, trafficking of immune cells, activation of key genes in host immunity to viral infection, and response to tissue damage. By utilizing luciferase enzymes with different emission spectra and/or substrates, it is possible to monitor two different biologic processes in the same animal, such as pathogen replication and sites of tissue injury. Combining imaging reporter viruses with genetically-engineered reporter mice is expected to substantially enhance the power of bioluminescence imaging for quantitative studies of viral and host factors that control disease outcome and effects of established and new therapeutic agents. PMID:20417377

  19. Kinetic study of trichloroethylene and toluene degradation by a bioluminescent reporter bacterium

    SciTech Connect

    Kelly, C.J.; Sanseverino, J.; Bienkowski, P.R.; Sayler, G.S.

    1995-12-31

    A constructed bioluminescent reporter bacterium, Pseudomonas putida B2, is very briefly described in this paper. The bacterium degrades toluene and trichloroethylene (TCE), and produces light in the presence of toluene. The light response is an indication of cellular viability and expression of the genes encoding toluene and TCE degrading enzymes.

  20. Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.

    PubMed

    Shimizu, Takeshi; Ohta, Yuko; Tsutsuki, Hiroyasu; Noda, Masatoshi

    2011-06-01

    A novel chromosome-plasmid hybrid bioluminescent reporter system (C-P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia coli (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C-P reporter system is useful for determining the gene expression profile of bacteria.

  1. An improved bioluminescence-based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor.

    PubMed

    Fukutani, Yosuke; Ishii, Jun; Noguchi, Keiichi; Kondo, Akihiko; Yohda, Masafumi

    2012-12-01

    The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein α-subunit (Gpa1p) with the olfactory-specific Gα(olf), the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast.

  2. Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage.

    PubMed

    Schofield, David A; Molineux, Ian J; Westwater, Caroline

    2012-08-01

    The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific 'bioluminescent' reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method.

  3. Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis.

    PubMed

    Damron, F Heath; McKenney, Elizabeth S; Barbier, Mariette; Liechti, George W; Schweizer, Herbert P; Goldberg, Joanna B

    2013-07-01

    We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

  4. Temperature-modulated bioluminescence tomography

    NASA Astrophysics Data System (ADS)

    Wang, Ge; Shen, Haiou; Cong, Wenxiang; Zhao, Shan; Wei, Guo Wei

    2006-08-01

    It was recently reported that bioluminescent spectra can be significantly affected by temperature, which we recognize as a major opportunity to overcome the inherent illposedness of bioluminescence tomography (BLT). In this paper, we propose temperature-modulated bioluminescence tomography (TBT) to utilize the temperature dependence of bioluminescence for superior BLT performance. Specifically, we employ a focused ultrasound array to heat small volumes of interest (VOI) one at a time, and induce a detectable change in the optical signal on the body surface of a mouse. Based on this type of information, the BLT reconstruction can be stabilized and improved. Our numerical experiments clearly demonstrate the merits of our TBT with either noise-free or noisy datasets. Also, this idea is applicable in 2D bioluminescence imaging and computational optical biopsy (COB). We believe that our approach and technology represents a major step forward in the field of BLT, and has an important and immediate applicability in bioluminescence imaging of small animals in general.

  5. Development of a novel, bioluminescence-based, fungal bioassay for toxicity testing.

    PubMed

    Weitz, Hedda J; Campbell, Colin D; Killham, Ken

    2002-07-01

    Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing. Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc. The results suggested that 60 min was a suitable exposure time for the bioassay. Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A. mellea and M. citricolor, but to Zn only for A. mellea. Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M. citricolor. The EC50 values for A. mellea and M. citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors. They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor. The results highlighted the importance of using both prokaryotic and eukaryotic biosensors. The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi. It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.

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

    PubMed

    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

  7. Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

    PubMed

    Cevenini, Luca; Calabretta, Maria Maddalena; Lopreside, Antonia; Tarantino, Giuseppe; Tassoni, Annalisa; Ferri, Maura; Roda, Aldo; Michelini, Elisa

    2016-12-01

    The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

  8. PCR-based detection of bioluminescent microbial populations in Tyrrhenian Sea

    NASA Astrophysics Data System (ADS)

    Gentile, Gabriela; De Luca, Massimo; Denaro, Renata; La Cono, Violetta; Smedile, Francesco; Scarfì, Simona; De Domenico, Emilio; De Domenico, Maria; Yakimov, Michail M.

    2009-05-01

    The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of l uxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750 m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in "compartment"-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, "yet to be cultivated" lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea.

  9. Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

    PubMed

    Schofield, David A; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2012-05-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

  10. Development of an Engineered Bioluminescent Reporter Phage for Detection of Bacterial Blight of Crucifers

    PubMed Central

    Bull, Carolee T.; Rubio, Isael; Wechter, W. Patrick; Westwater, Caroline; Molineux, Ian J.

    2012-01-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis. PMID:22427491

  11. Specific and Quantitative Assessment of Naphthalene and Salicylate Bioavailability by Using a Bioluminescent Catabolic Reporter Bacterium

    PubMed Central

    Heitzer, Armin; Webb, Oren F.; Thonnard, Janeen E.; Sayler, Gary S.

    1992-01-01

    A bioassay was developed and standardized for the rapid, specific, and quantitative assessment of naphthalene and salicylate bioavailability by use of bioluminescence monitoring of catabolic gene expression. The bioluminescent reporter strain Pseudomonas fluorescens HK44, which carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism, was used. The physiological state of the reporter cultures as well as the intrinsic regulatory properties of the naphthalene degradation operon must be taken into account to obtain a high specificity at low target substrate concentrations. Experiments have shown that the use of exponentially growing reporter cultures has advantages over the use of carbon-starved, resting cultures. In aqueous solutions for both substrates, naphthalene and salicylate, linear relationships between initial substrate concentration and bioluminescence response were found over concentration ranges of 1 to 2 orders of magnitude. Naphthalene could be detected at a concentration of 45 ppb. Studies conducted under defined conditions with extracts and slurries of experimentally contaminated sterile soils and identical uncontaminated soil controls demonstrated that this method can be used for specific and quantitative estimations of target pollutant presence and bioavailability in soil extracts and for specific and qualitative estimations of napthalene in soil slurries. PMID:16348717

  12. Specific and quantitative assessment of naphthalene and salicylate bioavailability by using a bioluminescent catabolic reporter bacterium

    SciTech Connect

    Heitzer, A.; Thonnard, J.E.; Sayler, G.S.; Webb, O.F. )

    1992-06-01

    A bioassay was developed and standardized for the rapid, specific, and quantitative assessment of naphthalene and salicylate bioavailability by use of bioluminescence monitoring of catabolic gene expression. The bioluminescent reporter strain Pseudomonas fluorescens HK44, which carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism, was used. The physiological state of the reporter cultures as well as the intrinsic regulatory properties of the naphthalene degradation operon must be taken into account to obtain a high specificity at low target substrate concentrations. Experiments have shown that the use of exponentially growing reporter cultures has advantages over the use of carbon-starved, resting cultures. In aqueous solutions for both substrates, naphthalene and salicylate, linear relationships between initial substrate concentration and bioluminescence response were found over concentration ranges of 1 to 2 orders of magnitude. Naphthalene could be detected at a concentration of 45 ppb. Studies conducted under defined conditions with extracts and slurries of experimentally contaminated sterile soils and identical uncontaminated soil controls demonstrated that this method can be used for specific and quantitative estimations of target pollutant presence and bioavailability in soil extracts and for specific and qualitative estimations of napthalene in soil slurries.

  13. Iterative method for bioluminescence tomography based on the radiative transport equation

    NASA Astrophysics Data System (ADS)

    Cong, Wenxiang; Wang, Ge

    2006-08-01

    Bioluminescence tomography (BLT) is a new molecular imaging modality, which helps study cancer and other diseases, develop drugs, and so on. This technology localizes and quantifies a bioluminescent source inside a living transgenic mouse, and is very useful in many biomedical applications. In this paper, we propose a novel algorithm based on the radiative transport equation to reconstruct the bioluminescence source distribution from data measured on the external surface of a mouse. Our approach transforms the transport equation into an integral equation of the second kind, and establishes a linear system to link the measured photon fluence rate with the unknown light source variables. A regularization measure is taken to overcome the ill-posedness of the inverse problem. Then, an iterative optimization technique with a simple constrain is employed to compute the desirable solution. The physical phantom experiments have been performed to demonstrate the feasibility of the reconstruction method, and evaluate its performance in terms of source location and power estimation.

  14. Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.

    PubMed

    Song, Ge; Wu, Qing-Ping; Xu, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Zhang, Shi-Fu; Guo, Zhan-Yun

    2015-12-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies.

  15. Mechanosensitivity of a Rapid Bioluminescence Reporter System Assessed by Atomic Force Microscopy

    PubMed Central

    Tesson, Benoit; Latz, Michael I.

    2015-01-01

    Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s−1, the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s−1, indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties. PMID:25809248

  16. Mechanosensitivity of a rapid bioluminescence reporter system assessed by atomic force microscopy.

    PubMed

    Tesson, Benoit; Latz, Michael I

    2015-03-24

    Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s(-1), the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s(-1), indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Three-dimensional multi bioluminescent sources reconstruction based on adaptive finite element method

    NASA Astrophysics Data System (ADS)

    Ma, Xibo; Tian, Jie; Zhang, Bo; Zhang, Xing; Xue, Zhenwen; Dong, Di; Han, Dong

    2011-03-01

    Among many optical molecular imaging modalities, bioluminescence imaging (BLI) has more and more wide application in tumor detection and evaluation of pharmacodynamics, toxicity, pharmacokinetics because of its noninvasive molecular and cellular level detection ability, high sensitivity and low cost in comparison with other imaging technologies. However, BLI can not present the accurate location and intensity of the inner bioluminescence sources such as in the bone, liver or lung etc. Bioluminescent tomography (BLT) shows its advantage in determining the bioluminescence source distribution inside a small animal or phantom. Considering the deficiency of two-dimensional imaging modality, we developed three-dimensional tomography to reconstruct the information of the bioluminescence source distribution in transgenic mOC-Luc mice bone with the boundary measured data. In this paper, to study the osteocalcin (OC) accumulation in transgenic mOC-Luc mice bone, a BLT reconstruction method based on multilevel adaptive finite element (FEM) algorithm was used for localizing and quantifying multi bioluminescence sources. Optical and anatomical information of the tissues are incorporated as a priori knowledge in this method, which can reduce the ill-posedness of BLT. The data was acquired by the dual modality BLT and Micro CT prototype system that was developed by us. Through temperature control and absolute intensity calibration, a relative accurate intensity can be calculated. The location of the OC accumulation was reconstructed, which was coherent with the principle of bone differentiation. This result also was testified by ex vivo experiment in the black 96-plate well using the BLI system and the chemiluminescence apparatus.

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

    PubMed

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

    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 have previously reported the ~2-fold enhanced activity and 1.4-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 femtomol levels of ATP. Additionally, 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 in living cells offering an improved alternative to Promega’s luc2 for reporter and imaging applications. PMID:26049097

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  2. Luminol-based bioluminescence imaging of mouse mammary tumors.

    PubMed

    Alshetaiwi, Hamad S; Balivada, Sivasai; Shrestha, Tej B; Pyle, Marla; Basel, Matthew T; Bossmann, Stefan H; Troyer, Deryl L

    2013-10-05

    Polymorphonuclear neutrophils (PMNs) are the most abundant circulating blood leukocytes. They are part of the innate immune system and provide a first line of defense by migrating toward areas of inflammation in response to chemical signals released from the site. Some solid tumors, such as breast cancer, also cause recruitment and activation of PMNs and release of myeloperoxidase. In this study, we demonstrate that administration of luminol to mice that have been transplanted with 4T1 mammary tumor cells permits the detection of myeloperoxidase activity, and consequently, the location of the tumor. Luminol allowed detection of activated PMNs only two days after cancer cell transplantation, even though tumors were not yet palpable. In conclusion, luminol-bioluminescence imaging (BLI) can provide a pathway towards detection of solid tumors at an early stage in preclinical tumor models. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Bioengineered bioluminescent magnetotactic bacteria as a powerful tool for chip-based whole-cell biosensors.

    PubMed

    Roda, Aldo; Cevenini, Luca; Borg, Sarah; Michelini, Elisa; Calabretta, Maria Maddalena; Schüler, Dirk

    2013-12-21

    This paper describes the generation of genetically engineered bioluminescent magnetotactic bacteria (BL-MTB) and their integration into a microfluidic analytical device to create a portable toxicity detection system. Magnetospirillum gryphiswaldense strain MSR-1 was bioengineered to constitutively express a red-emitting click beetle luciferase whose bioluminescent signal is directly proportional to bacterial viability. The magnetic properties of these bacteria have been exploited as "natural actuators" to transfer the cells in the chip from the reaction to the detection area, optimizing the chip's analytical performance. A robust and cost-effective biosensor for the evaluation of sample toxicity, named MAGNETOX, based on lens-free contact imaging detection, has been developed. A microfluidic chip has been fabricated using multilayered black and transparent polydimethyl siloxane (PDMS) in which BL-MTB are incubated for 30 min with the sample, then moved by microfluidics, trapped, and concentrated in detection chambers by an array of neodymium-iron-boron magnets. The chip is placed in contact with a cooled CCD via a fiber optic taper to perform quantitative bioluminescence imaging after addition of luciferin substrate. A model toxic compound (dimethyl sulfoxide, DMSO) and a bile acid (taurochenodeoxycholic acid, TCDCA) were used to investigate the analytical performance of the MAGNETOX. Incubation with DMSO and TCDCA drastically reduces the bioluminescent signal in a dose-related manner. The generation of bacteria that are both magnetic and bioluminescent combines the advantages of easy 2D cell handling with ultra sensitive detection, offering undoubted potential to develop cell-based biosensors integrated into microfluidic chips.

  4. 'Bioluminescent' reporter phage for the detection of Category A bacterial pathogens.

    PubMed

    Schofield, David A; Molineux, Ian J; Westwater, Caroline

    2011-07-08

    Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens. For demonstration purposes, here we describe the method for the phage

  5. A bioluminescence reporter mouse that monitors expression of constitutively active β-catenin.

    PubMed

    Szwarc, Maria M; Kommagani, Ramakrishna; Peavey, Mary C; Hai, Lan; Lonard, David M; Lydon, John P

    2017-01-01

    This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse-when crossed with the appropriate effector transgenic-will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues.

  6. A bioluminescence reporter mouse that monitors expression of constitutively active β-catenin

    PubMed Central

    Kommagani, Ramakrishna; Peavey, Mary C.; Hai, Lan; Lonard, David M.; Lydon, John P.

    2017-01-01

    This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse—when crossed with the appropriate effector transgenic—will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues. PMID:28253313

  7. Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage

    PubMed Central

    Schofield, David A.; Molineux, Ian J.; Westwater, Caroline

    2012-01-01

    The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific ‘bioluminescent’ reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method. PMID:22579583

  8. Bacillus anthracis diagnostic detection and rapid antibiotic susceptibility determination using 'bioluminescent' reporter phage.

    PubMed

    Schofield, David A; Sharp, Natasha J; Vandamm, Joshua; Molineux, Ian J; Spreng, Krista A; Rajanna, Chythanya; Westwater, Caroline; Stewart, George C

    2013-11-01

    Genetically modified phages have the potential to detect pathogenic bacteria from clinical, environmental, or food-related sources. Herein we assess an engineered 'bioluminescent' reporter phage (Wß::luxAB) as a clinical diagnostic tool for Bacillus anthracis, the etiological agent of anthrax. Wß::luxAB is able to rapidly (within minutes) detect a panel of B. anthracis strains by transducing a bioluminescent phenotype. The reporter phage displays species specificity by its inability, or significantly reduced ability, to detect members of the closely related Bacillus cereus group and other common bacterial pathogens. Using spiked clinical specimens, Wß::luxAB detects B. anthracis within 5 h at clinically relevant concentrations, and provides antibiotic susceptibility information that mirrors the CLSI method, except that data are obtained at least 5-fold faster. Although anthrax is a treatable disease, a positive patient prognosis is dependent on timely diagnosis and appropriate therapy. Wß::luxAB rapidly detects B. anthracis and determines antibiotic efficacy, properties that will help patient outcome.

  9. Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    PubMed

    Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

    2015-11-01

    Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  11. Development of a rapid optic bacteria detecting system based on ATP bioluminescence

    NASA Astrophysics Data System (ADS)

    Liu, Jun Tao; Luo, JinPing; Liu, XiaoHong; Cai, XinXia

    2014-12-01

    A rapid optic bacteria detecting system based on the principle of Adenosine triphosphate(ATP) bioluminescence was presented in this paper. This system consisted of bioluminescence-based biosensor and the high-sensitivity optic meter. A photon counting photomultiplier tube (PMT) module was used to improve the detection sensitivity, and a NIOS II/f processor based on a Field Programmable Gate Array(FPGA) was used to control the system. In this work, Micrococcus luteus were chosen as the test sample. Several Micrococcus luteus suspension with different concentration was tested by both T2011 and plate counting method. By comparing the two group results, an calibration curve was obtained from the bioluminescence intensity for Micrococcus luteus in the range of 2.3×102 ~ 2.3×106 CFU/mL with a good correlation coefficient of 0.960. An impacting Air microorganism sampler was used to capture Airborne Bacteria, and 8 samples were collected in different place. The TBC results of 8 samples by T2011 were between 10 ~ 2×103 cfu/mL, consistent with that of plate counting method, which indicated that 8 samples were between 10 ~ 3×103 cfu/mL. For total airborne bacteria count was small, correlation coefficient was poor. Also no significant difference was found between T2011 and plate counting method by statistical analyses.

  12. Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

    PubMed

    Gorokhovatsky, Andrey Yu; Rudenko, Natalia V; Marchenkov, Victor V; Skosyrev, Vitaly S; Arzhanov, Maxim A; Burkhardt, Nils; Zakharov, Mikhail V; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2003-02-01

    Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

  13. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    PubMed

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  14. In vivo bioluminescence tomography based on multi-view projection and 3D surface reconstruction

    NASA Astrophysics Data System (ADS)

    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.

  15. Study of effectiveness of bioluminescent reporter phage assay on Y. pseudotuberculosis strains.

    PubMed

    Mitiashvili, M R

    2013-05-01

    The method describes the phage-mediated transduction of a bioluminescent phenotype to cultivated Y. pseudotuberculosis cells which are subsequently measured using a microplate luminometer. Reporter phage assay is rapid detection technique and its efficiency is not affected by presence of contaminating bacteria, no sample preparation is needed and it has the ability to test multiple samples simultaneously in a 96-well microtiter plate format. Experiments were performed to develop the rapid detection technique for Y. pseudotuberculosis strains and study the ability of a reporter Yersinia phage to confer a bioluminescent signal to Y. pseudotuberculosis strains under different environmental conditions (media, temperature, bacterial number) for detection. Further, to determine if the Yersinia phage can detect Y. pseudotuberculosis in presence of other bacterial species. The results revealed that the developed reporter phage assay is not effective against wide range of Y. pseudotuberculosis. Y. pseudotuberculosis could be rapidly detected within 30 minutes at 28°C. The reporter phage assay could detect luminescence within 45 minutes when the bacterial cells were at the minimal concentration 105 cells/mL. The optimal detectable concentrations were 106-107 cells/mL at 28 and 37°C. The reporter phage assay could detect Y. pseudotuberculosis within 30 minutes in presence of other enteric bacteria without selective enrichment. It should be noted that the Yersinia reporter phage is specific to Yersinia pestis strains and it can be used to detect Y. pseudotuberculosis when samples exclude the existence of Y. pestis strains. In the presented study this aspect was foreseen.

  16. Bioluminescence Resonance Energy Transfer (BRET)-Based Synthetic Sensor Platform for Drug Discovery.

    PubMed

    Woo, Jongchan; Hong, Jason; Dinesh-Kumar, Savithramma P

    2017-04-03

    Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein-protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro. Here, we report the methods for constructing a biosensor of human HsLC3a as a probe for autophagy biogenesis and the optimization of the intramolecular BRET assay that allows for high-throughput screening of chemical modulators of autophagy. User-friendly working interface with the BRET-based synthetic sensor of HsLC3a makes drug discovery easy and amenable for high-throughput. The BRET protocol described here could be easily applicable to generate other biosensors for monitoring PPIs by measurement of intermolecular BRET. © 2017 by John Wiley & Sons, Inc.

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

    PubMed

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

    2009-11-01

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

  18. Flow injection analysis with bioluminescence-based fiber-optic biosensors

    NASA Astrophysics Data System (ADS)

    Blum, Loic J.; Gautier, Sabine; Coulet, Pierre R.

    1991-09-01

    Fiber optic biosensors based on the firefly and the bacterial bioluminescence reactions have been constructed and incorporated in a specially designed flow-cell for the sensitive determination of ATP and NADH, respectively. The bioluminescence enzymes were immobilized on preactivated polyamide membranes which were placed in close contact with the surface on one end of a glass-fiber bundle, the other end being connected to the photomultiplier tube of a luminometer. When using the continuous-flow device with the firefly luciferase or the bacterial system immobilized separately on different membranes, the detection limit for ATP and NADH were 0.25 and 2 pmol, respectively. The versatility of the fiber optic probe has been improved by co-immobilizing the bacterial bioluminescent system and the firefly luciferase on the same support enabling the use of a single sensor for the selective, specific, and alternate determination of these two analytes. Compatible reaction conditions preserving the activity of each co-immobilized enzyme without impairing its stability were found. The selection of the appropriate reaction medium was done using a four port valve. Alternate quantification of ATP and NADH could then be performed in the linear ranges 0.25 pmol - 3 nmol and 5 pmol - 1 nmol, respectively with a RSD of 4.0 - 4.5%.

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

    PubMed Central

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

    2014-01-01

    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. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated Click Beetle-(cLuc), Firefly-(fLuc), Renilla-(rLuc), and Gaussia-(gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC-transporters (ABCB1, ABCC1 and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact-cells compared to cell-lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC-transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity >2-fold in HEK-293/cLuc, fLuc and rLuc cells; inhibition of ABCB1 elevated the BLI intensity 2-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC-transporter/luciferase-reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates D-luciferin-based BLI in a luciferase-type-independent manner. Little ABC-transporter effect on gLuc-BLI intensity is observed since a large fraction of gLuc is secreted. The expression level of ABC-transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research. PMID:21496450

  20. The use of a genetically engineered Pseudomonas species (Shk1) as a bioluminescent reporter for heavy metal toxicity screening in wastewater treatment plant influent.

    PubMed

    Ren, Shijin; Frymier, Paul D

    2003-01-01

    Heavy metals are known to be inhibitory and toxic to the activated-sludge microbial community in biological wastewater treatment plants. Toxicity screening of aqueous mixtures of these heavy metal ions in plant influent could use both chemical and biological methods. As a biological method, luminescent bacterial bioreporters offer the advantages of a simple test procedure and rapid response. Current biologically based methods for screening aqueous streams for toxicity are labor-intensive, inaccurate, or difficult to use in continuous monitoring applications. In the present study, a system was developed that is simple and easily automated. This system is based on the bacterium Shk1, a genetically engineered bioluminescent Pseudomonad whose parent strain was originally isolated from activated sludge. Compared with other bioluminescence-based systems (specifically, the Microtox assay), the system of the present study more accurately reflects the effects of the toxicity of common metal ions on activated-sludge respirometry without being overly sensitive to typical constituents of wastewater. The use of Shk1 as a bioluminescent reporter for heavy metal toxicity testing for the application of wastewater treatment influent toxicity screening is presented in this study.

  1. Evaluation of the ecotoxicity of pollutants with bioluminescent microorganisms.

    PubMed

    Fernández-Piñas, Francisca; Rodea-Palomares, Ismael; Leganés, Francisco; González-Pleiter, Miguel; Angeles Muñoz-Martín, M

    2014-01-01

    This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.

  2. Influence of Antibiotic Pressure on Five Plasmid-based Bioluminescent Gram-negative Bacterial Strains.

    PubMed

    Wang, Xiwen; Chi, Hang; Li, Qianxue; Li, Wenliang; Li, Jiakuan; Li, Bo; Gao, Weicun; Zhang, Da; Sun, Yu; Yi, Le; Qu, Han; Wang, Yutian; Li, Zhiping; Xia, Zhiping

    2017-08-08

    The present study aims to develop five Gram-negative bacteria expressing bacterial luciferase for use to evaluate the influence of different antibiotics on bacterial bioluminescence. The pBBR-lux plasmid was introduced into five Gram-negative bacteria; the bioluminescent signals and colony-forming unit (CFU)/ml of all the bioluminescent strains were monitored with six antibiotics at various concentrations. Dose-dependent bioluminescence signals can be used for rapid bacterial antibiotic susceptibility test (AST). All five bioluminescent bacterial strains have similar bioluminescence and CFU enhancement at sub-minimum inhibitory concentration (MIC) of six different antibiotics. The bioluminescent signals and CFU enhancement at sub-MIC antibiotic concentrations should be of value in the research of new antibiotic drugs and bioluminescent imaging.

  3. Construction of bioluminescent cyanobacterial reporter strains for detection of nickel, cobalt and zinc.

    PubMed

    Peca, Loredana; Kós, Péter B; Máté, Zoltan; Farsang, Andrea; Vass, Imre

    2008-12-01

    Two whole-cell bioluminescent reporters were constructed by fusing the reporter genes luxAB with the Co(2+) and Zn(2+) inducible coaT promoter or the Ni(2+)-inducible nrsBACD promoter, respectively, in the genome of Synechocystis sp. PCC 6803. The obtained reporters, designated coaLux and nrsLux, respectively, responded quantitatively to metal ions. After 3 h incubation at 40 micromol m(-2) s(-1) visible light, the detection range of coaLux was 0.3-6 microM for Co(2+) and 1-3 microM for Zn(2+). Incubation in darkness increased the detection range by about four times. The nrsLux reporter was specific to Ni(2+), with a detection range of 0.2-6 microM. However, its activity was inhibited by Zn(2+) with a half maximal inhibitory concentration c. 6 microM, and totally inhibited by darkness. This is the first whole-cell Ni(2+)-specific reporter with a clear dose-signal relationship. In a soil-like mixture of different chemical and oil industry wastes, the coaLux reporter strain detected about 90% of the zinc content of the sample. This study demonstrates the potential for development of a rapid, simple and economical field assay for nickel, cobalt and zinc detection using the coaLux and nrsLux reporters.

  4. A measurement-based analytical approach to the bioluminescence tomography problem

    NASA Astrophysics Data System (ADS)

    Erkol, Hakan; Demirkiran, Aytac; Kipergil, Esra-Aytac; Uluc, Nasire; Unlu, Mehmet B.

    2014-03-01

    This work presents an analytical approach for the solution of the tissue diffusion equation based on the bound- ary measurements. We consider a bioluminescent point source in both homogeneous and heterogeneous circular turbid media. The point source is described by the Dirac delta function. Analytical expressions for the strength and position of the point source are obtained introducing boundary measurements and then applying appropriate boundary conditions. In addition, numerical simulations are performed for the position of the source. Calculations show that that the analytical results are in a good accordance with the numerical results.

  5. Online monitoring of water toxicity by use of bioluminescent reporter bacterial biochips.

    PubMed

    Elad, Tal; Almog, Ronen; Yagur-Kroll, Sharon; Levkov, Klimentiy; Melamed, Sahar; Shacham-Diamand, Yosi; Belkin, Shimshon

    2011-10-01

    We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.

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

    PubMed

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

    2016-01-08

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

  7. Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter.

    PubMed

    Goya, María Eugenia; Romanowski, Andrés; Caldart, Carlos S; Bénard, Claire Y; Golombek, Diego A

    2016-11-29

    Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems-including mice, flies, fungi, plants, and bacteria-have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.

  8. Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter

    PubMed Central

    Goya, María Eugenia; Romanowski, Andrés; Caldart, Carlos S.; Bénard, Claire Y.; Golombek, Diego A.

    2016-01-01

    Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems—including mice, flies, fungi, plants, and bacteria—have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C. elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C. elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C. elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode. PMID:27849618

  9. Bioluminescence patterns among North American Armillaria species.

    PubMed

    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.

  10. Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.

    PubMed

    Branchini, Bruce R; Rosenberg, Justin C; Ablamsky, Danielle M; Taylor, Kelsey P; Southworth, Tara L; Linder, Samantha J

    2011-07-15

    We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2011-01-01

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

  12. Bioluminescence-based method for measuring assimilable organic carbon in pretreatment water for reverse osmosis membrane desalination.

    PubMed

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

    2011-02-01

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

  13. Comparison of two quantum dots for bioluminescence resonance energy transfer based on nucleic acid detection

    NASA Astrophysics Data System (ADS)

    Zhang, Daohong

    2017-05-01

    The performance of two commercially available quantum dots, quantum dot 605 (Qd605) and quantum dot 625 (Qd625), was tested and compared in the sensing system developed by our group previously. The sandwich format sensing system employed Renilla luciferase (Rluc) and quantum dots (Qds), could report the presence of targets with increasing bioluminescent resonance energy transfer (BRET) signal. The best spacing between the Rluc and Qds probes were 15 nucleotides. Both of Qd605 and Qd625 sensing system could quantify nucleic acid targets through 1-min hybridization from 0.2 picomoles. However, the Qd625 system showed higher BRET signal and better selectivity. Therefore, Qd625 is a better choice in this system compared to Qd605.

  14. L1/2 regularization based numerical method for effective reconstruction of bioluminescence tomography

    NASA Astrophysics Data System (ADS)

    Chen, Xueli; Yang, Defu; Zhang, Qitan; Liang, Jimin

    2014-05-01

    Even though bioluminescence tomography (BLT) exhibits significant potential and wide applications in macroscopic imaging of small animals in vivo, the inverse reconstruction is still a tough problem that has plagued researchers in a related area. The ill-posedness of inverse reconstruction arises from insufficient measurements and modeling errors, so that the inverse reconstruction cannot be solved directly. In this study, an l1/2 regularization based numerical method was developed for effective reconstruction of BLT. In the method, the inverse reconstruction of BLT was constrained into an l1/2 regularization problem, and then the weighted interior-point algorithm (WIPA) was applied to solve the problem through transforming it into obtaining the solution of a series of l1 regularizers. The feasibility and effectiveness of the proposed method were demonstrated with numerical simulations on a digital mouse. Stability verification experiments further illustrated the robustness of the proposed method for different levels of Gaussian noise.

  15. Comparative studies of lp-regularization-based reconstruction algorithms for bioluminescence tomography

    PubMed Central

    Zhang, Qitan; Chen, Xueli; Qu, Xiaochao; Liang, Jimin; Tian, Jie

    2012-01-01

    Inverse source reconstruction is the most challenging aspect of bioluminescence tomography (BLT) because of its ill-posedness. Although many efforts have been devoted to this problem, so far, there is no generally accepted method. Due to the ill-posedness property of the BLT inverse problem, the regularization method plays an important role in the inverse reconstruction. In this paper, six reconstruction algorithms based on lp regularization are surveyed. The effects of the permissible source region, measurement noise, optical properties, tissue specificity and source locations on the performance of the reconstruction algorithms are investigated using a series of single source experiments. In order to further inspect the performance of the reconstruction algorithms, we present the double sources and the in vivo mouse experiments to study their resolution ability and potential for a practical heterogeneous mouse experiment. It is hoped to provide useful guidance on algorithm development and application in the related fields. PMID:23162729

  16. Basic and Applied Aspects of Color Tuning of Bioluminescence Systems

    NASA Astrophysics Data System (ADS)

    Ohmiya, Yoshihiro

    2005-09-01

    V. Viviani et al. [Biochemistry 38 (1999) 8271] were the first to succeed in cloning the red-emitting enzyme from the South American railroad worm, which is the only bioluminescent organism known to emit a red-colored light. The application of red bioluminescence has been our goal because the transmittance of longer-wavelength light is superior to that of the other colors for visualization of biological functions in living cells. Now, different color luciferases, which emit with wavelength maxima ranging from 400 to 630 nm, are available and are being used. For example, based on different color luciferases, Nakajima et al. developed a tricolor reporter in vitro assay system based on these different color luciferases in which the expression of three genes can be monitored simultaneously. On the other hand, bioluminescence resonance energy transfer (BRET) is a natural phenomenon caused by the intermolecular interaction between a bioluminescent protein and a fluorophore on a second protein, resulting in the light from the bioluminescence reaction having the spectrum of the fluorophore. Otsuji et al. [Anal. Biochem. 329 (2004) 230] showed that the change in the efficiency of energy transfer in intramolecular BRET can quantify cellular functions in living cells. In this review, I introduce the basic mechanisms of color tuning in bioluminescent systems and new applications based on color tuning in the life sciences.

  17. Destabilized bioluminescent proteins

    DOEpatents

    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.

  18. Cell-based galactosemia diagnosis system based on a galactose assay using a bioluminescent Escherichia coli array.

    PubMed

    Woo, Min-Ah; Kim, Moon Il; Cho, Daeyeon; Park, Hyun Gyu

    2013-11-19

    A new cell-based galactose assay system, which is comprised of two bioluminescent Escherichia coli strains immobilized within an agarose gel arrayed on a well plate, has been developed. For this purpose, a galT knockout strain [galT(-) cell] of E. coli was genetically constructed so that cell growth is not promoted by galactose but rather by glucose present in a sample. Another E. coli W strain (normal cell), which grows normally in the presence of either glucose or galactose, was employed. A luminescent reporter gene, which produces luminescence as cells grow, was inserted into both of the E. coli strains, so that cell growth could be monitored in a facile manner. The two strains were separately grown for 4 h on gel arrays to which test samples were individually supplied. The relative luminescence unit (RLU) values caused by cell growth were determined for each array, one of which is resulted by glucose only and the other of which is resulted by both glucose and galactose present in the sample. By employing this protocol, galactose concentrations present in the test sample are reflected in the differences between the RLU values for each array. The practical utility of the new assay system was demonstrated by its use in determining galactose levels in clinical blood spot specimens coming from newborn babies. Because it can be employed to diagnosis of galactosemia in newborn babies in a more rapid, convenient, and cost-effective manner, this cell-based solid-phase galactose assay system should become a powerful alternative to conventional methods, which require labor-intensive and time-consuming procedures and/or complicated and expensive equipment.

  19. Hybrid radiosity-SP3 equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    NASA Astrophysics Data System (ADS)

    Chen, Xueli; Zhang, Qitan; Yang, Defu; Liang, Jimin

    2014-01-01

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP3 equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP3) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  20. Hepatocellular Carcinoma Cells Carrying a Multimodality Reporter Gene for Fluorescence, Bioluminescence, and Magnetic Resonance Imaging In Vitro and In Vivo.

    PubMed

    Qin, Xiaoxiao; Hu, Xiaojun; Wu, Chun; Cai, Mingyue; Li, Zhengran; Zhang, Lina; Lin, Liteng; Huang, Wensou; Zhu, Kangshun

    2016-11-01

    The study aimed to evaluate the feasibility of imaging or tracking hepatocellular carcinoma cells by modifying these cells to carry a multimodality reporter gene, enabling fluorescence, bioluminescence, and magnetic resonance imaging (MRI) in vitro and in vivo. HepG2 cells were labeled with the enhanced green fluorescent protein (EGFP)/luciferase2/ferritin-the multimodality reporter gene (labeled HepG2 cells). The labeled and unlabeled HepG2 cells were cultured in vitro and then injected subcutaneously into mice as a hepatoma model in vivo. The expressions of EGFP, luciferase2, and ferritin in HepG2 cell suspensions and hepatoma model were investigated using fluorescence, bioluminescence, and MRI. Individual HepG2 cells expressing EGFP were identified under blue laser excitation. The linear coefficient between the optical signal intensity of luciferase2 and the number of labeled cells was 0.993. MRI was used to distinguish the T2* signal of 2 × 10(7) cells/mL between the labeled (6.67 ± 1.88 ms) and unlabeled cells (10.66 ± 2.22 ms) (P = 0.034). In vivo, individual HepG2 cells expressing EGFP in frozen sections were observed. Labeled cells expressing luciferase2 have been detected since the second day after injection, and the bioluminescence increased with the tumor size. The T2* signal was significantly different between the labeled (6.04 ± 1.60 ms) and unlabeled cells (17.06 ± 2.17 ms) (P <0.001). A multimodality reporter gene consisting of EGFP, luciferase2, and ferritin was successfully integrated into the HepG2 cell genome via a lentiviral vector and was highly expressed in the daughter cells. These cells could be detected by fluorescence, bioluminescence, and MRI in vitro and in vivo. Copyright © 2016 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.

  1. Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease.

    PubMed

    Li, Fengyun; Yu, Junping; Zhang, Zhiping; Cui, Zongqiang; Wang, Dianbing; Wei, Hongping; Zhang, Xian-En

    2012-04-29

    Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

  2. The CUBLAS and CULA based GPU acceleration of adaptive finite element framework for bioluminescence tomography.

    PubMed

    Zhang, Bo; Yang, Xiang; Yang, Fei; Yang, Xin; Qin, Chenghu; Han, Dong; Ma, Xibo; Liu, Kai; Tian, Jie

    2010-09-13

    In molecular imaging (MI), especially the optical molecular imaging, bioluminescence tomography (BLT) emerges as an effective imaging modality for small animal imaging. The finite element methods (FEMs), especially the adaptive finite element (AFE) framework, play an important role in BLT. The processing speed of the FEMs and the AFE framework still needs to be improved, although the multi-thread CPU technology and the multi CPU technology have already been applied. In this paper, we for the first time introduce a new kind of acceleration technology to accelerate the AFE framework for BLT, using the graphics processing unit (GPU). Besides the processing speed, the GPU technology can get a balance between the cost and performance. The CUBLAS and CULA are two main important and powerful libraries for programming on NVIDIA GPUs. With the help of CUBLAS and CULA, it is easy to code on NVIDIA GPU and there is no need to worry about the details about the hardware environment of a specific GPU. The numerical experiments are designed to show the necessity, effect and application of the proposed CUBLAS and CULA based GPU acceleration. From the results of the experiments, we can reach the conclusion that the proposed CUBLAS and CULA based GPU acceleration method can improve the processing speed of the AFE framework very much while getting a balance between cost and performance.

  3. Bioluminescence-based detection of microRNA, miR21 in breast cancer cells.

    PubMed

    Cissell, Kyle A; Rahimi, Yasmeen; Shrestha, Suresh; Hunt, Eric A; Deo, Sapna K

    2008-04-01

    A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer

  4. A method for atlas-based volumetric registration with surface constraints for optical bioluminescence tomography in small animal imaging

    NASA Astrophysics Data System (ADS)

    Chaudhari, Abhijit J.; Joshi, Anand A.; Darvas, Felix; Leahy, Richard M.

    2007-03-01

    Atlases are normalized representations of anatomy that can provide a standard coordinate system for in vivo imaging studies. For Optical Bioluminescence Tomography (OBT) in small animals, the animal's surface topography can be reconstructed from structured light measurements, but internal anatomy is unavailable unless additional CT or MR images are acquired. We present a novel method for estimating the internal organ structure of a mouse by warping a labeled 3D volumetric mouse atlas with the constraint that the surfaces of the two should match. Surface-constrained harmonic maps used for this bijective warping are computed by minimizing the covariant harmonic energy. We demonstrate the application of this warping scheme in OBT, where scattering and absorption coefficients of tissue are functions of the internal anatomy and hence, better estimates of the organ structures can lead to a more accurate forward model resulting in improved source localization. We first estimated the subject's internal geometry using the atlas-based warping scheme. Then the mouse was tessellated and optical properties were assigned based on the estimated organ structure. Bioluminescent sources were simulated, an optical forward model was computed using a finite-element solver, and multispectral data were simulated. We evaluate the accuracy of the forward model computed using the warped atlas against that assuming a homogeneous mouse model. This is done by comparing each model against a 'true' optical forward model where the anatomy of the mouse is assumed known. We also evaluate the impact of anatomical alignment on bioluminescence source localization.

  5. Bioluminescence imaging in live cells and animals

    PubMed Central

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

    2016-01-01

    Abstract. 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. PMID:27226972

  6. Bioluminescence imaging in live cells and animals.

    PubMed

    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.

  7. A lower limit of detection for atrazine was obtained using bioluminescent reporter bacteria via a lower incubation temperature.

    PubMed

    Jia, Kun; Eltzov, Evgeni; Toury, Timothée; Marks, Robert S; Ionescu, Rodica E

    2012-10-01

    The present article reports on the influence of various atrazine concentrations to the response of genetically modified Escherichia coli TV1061 bacterial cells while modulating the experimental conditions. Interesting increases of bioluminescence signals are recorded for E. coli TV1061 bacteria in the presence of 10 μg/mL atrazine concentration named "high-toxicity bacteria alert" when compared with 1 μg/mL -10 fg/mL atrazine termed "low-toxicity bacteria alert". Detecting the effect of atrazine via its effect on bioluminescence of bacteria has been carried out by two consecutive measurements (fresh and overnight modes) at different concentrations of analyte. We have shown that a more precise discrimination at lower-toxicity concentrations can be obtained through overnight incubation of bacteria with the analyte at 4 °C. In addition, centrifugation of bacterial cells and analyte dilutions has been performed in order to ensure a better interaction between the insoluble atrazine pesticide and the bacterial cells.

  8. L{sub 1/2} regularization based numerical method for effective reconstruction of bioluminescence tomography

    SciTech Connect

    Chen, Xueli E-mail: jimleung@mail.xidian.edu.cn; Yang, Defu; Zhang, Qitan; Liang, Jimin E-mail: jimleung@mail.xidian.edu.cn

    2014-05-14

    Even though bioluminescence tomography (BLT) exhibits significant potential and wide applications in macroscopic imaging of small animals in vivo, the inverse reconstruction is still a tough problem that has plagued researchers in a related area. The ill-posedness of inverse reconstruction arises from insufficient measurements and modeling errors, so that the inverse reconstruction cannot be solved directly. In this study, an l{sub 1/2} regularization based numerical method was developed for effective reconstruction of BLT. In the method, the inverse reconstruction of BLT was constrained into an l{sub 1/2} regularization problem, and then the weighted interior-point algorithm (WIPA) was applied to solve the problem through transforming it into obtaining the solution of a series of l{sub 1} regularizers. The feasibility and effectiveness of the proposed method were demonstrated with numerical simulations on a digital mouse. Stability verification experiments further illustrated the robustness of the proposed method for different levels of Gaussian noise.

  9. The uses and abuses of rapid bioluminescence-based ATP assays.

    PubMed

    Shama, G; Malik, D J

    2013-03-01

    Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens.

  10. A validated bioluminescence-based assay for the rapid determination of the initial rate of kill for discovery antimalarials.

    PubMed

    Ullah, Imran; Sharma, Raman; Biagini, Giancarlo A; Horrocks, Paul

    2017-03-01

    A future treatment for uncomplicated malaria will contain at least one component that exerts a rapid rate of kill. We describe here the validation and application of a simple, robust and rapid bioluminescence-based assay for the determination of the initial rate of kill in intra-erythrocytic asexual stages of Plasmodium falciparum . A modification to the concentration-response bioluminescence [here termed bioluminescence relative rate of kill (BRRoK)] assay, utilizing exposure to fold-IC 50 concentrations (0.33× to  9×), was used to monitor the immediate cytocidal effect of 372 open-source compounds for antimalarial drug discovery available through the Medicines for Malaria Venture Malaria Box. Antimalarial drugs that exert a rapid cytocidal effect produce a concentration-dependent loss of bioluminescence signal that correlates with available in vitro and in vivo estimates of parasite clearance time and parasite reduction ratio. Following the measurement of IC 50 for the Malaria Box compounds in Dd2 luc , the BRRoK assay was used to identify and rank 372 compounds for their initial cytocidal activity. Fifty-three compounds in the Malaria Box show an initial relative rate of kill greater than that of chloroquine, with 17 of these having an initial relative rate of kill greater than that of dihydroartemisinin. The BRRoK assay provides a rapid assay format for the estimation of a key pharmacodynamic property of antimalarial drug action. The simplicity and robustness of the assay suggests it would be readily scalable for high-throughput screening and a critical decision-making tool for antimalarial drug development.

  11. Molecular probes and bioluminescent reporters in ecological optimization of biodegradation. (FY 91 aasert). Annual report, 1 June 1993-31 May 1994

    SciTech Connect

    Sayler, G.S.

    1994-05-31

    The goal of the research supported by this grant is to determine the role that biosurfactants and synthetic surfactants play in enhancing the bioavailability of sorbed or immiscible-phase aromatic hydrocarbons (PAHs.) in particulate media. Increased bioavailability is assessed in terms of increased PAH-degrader population densities (nah gene frequencies) and their activities including the rate and/or extent of biodegradation and degradative gene expression as measured by bioluminescence response and mRNA levels. To achieve the proposed goal, bacterial strains containing specific degradative genes and bioluminescent reporter systems are being used to monitor the effectiveness of surfactants for enhancing the biodegradation of aromatic hydrocarbon contaminants in environmental simulations. These genetic marker systems allow for the quantitation of degradative gene frequency and activity. Construction of an improved bioluminescent reporter strain for PAH degradation is currently underway. This approach involves incorporation of a transposon containing the lower naphthalene pathway promoter fused to the lux genes (nah-lux) into the bacterial chromosome resulting in a stable gene fusion present as a single copy per cell.

  12. Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

    PubMed

    Branchini, Bruce

    2016-01-01

    We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.

  13. Microbiological Assay Using Bioluminescent Organism.

    DTIC Science & Technology

    A microbiological assay based on bioluminesce employing the bioluminescent dinoflagellate Pyrocystis lunula. An oil well drilling fluid sample is... Pyrocystis lunula in suspension. The mixture is agitated to subject th Pyrocystis lunula to a shear stress. Light emitted as a result of the shear...stress on the Pyrocystic lunula is measure and compared with a control to determine if there is diminution of light produced by the Pyrocystis lunula in

  14. Bioluminescence based biosensors for quantitative detection of enterococcal peptide-pheromone activity reveal inter-strain telesensing in vivo during polymicrobial systemic infection.

    PubMed

    La Rosa, Sabina Leanti; Solheim, Margrete; Diep, Dzung B; Nes, Ingolf F; Brede, Dag Anders

    2015-02-09

    Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants. This bacterium uses quorum-sensing systems to regulate its physiological processes, including the expression of virulence traits, to adapt and proliferate within a host. Here, we describe the construction and application of two bioluminescence-based reporter systems for the direct detection of the quorum-sensing regulated expression of (i) the gelatinase biosynthesis-activating pheromone (GBAP) and (ii) the cytolysin small subunit (CylL(S)) in natural samples. The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylL(S) both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity. Biosensors employed to investigate the interaction between the fsr and cyl systems revealed that fsr impeded CylL(S) activity by 75%. Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylL(S)-producers in its environment. This isolate enhanced its virulence during polymicrobial systemic infection of Galleria mellonella.

  15. Bioluminescence based biosensors for quantitative detection of enterococcal peptide–pheromone activity reveal inter-strain telesensing in vivo during polymicrobial systemic infection

    PubMed Central

    La Rosa, Sabina Leanti; Solheim, Margrete; Diep, Dzung B.; Nes, Ingolf F.; Brede, Dag Anders

    2015-01-01

    Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants. This bacterium uses quorum-sensing systems to regulate its physiological processes, including the expression of virulence traits, to adapt and proliferate within a host. Here, we describe the construction and application of two bioluminescence-based reporter systems for the direct detection of the quorum-sensing regulated expression of (i) the gelatinase biosynthesis-activating pheromone (GBAP) and (ii) the cytolysin small subunit (CylLS) in natural samples. The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylLS both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity. Biosensors employed to investigate the interaction between the fsr and cyl systems revealed that fsr impeded CylLS activity by 75%. Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylLS-producers in its environment. This isolate enhanced its virulence during polymicrobial systemic infection of Galleria mellonella. PMID:25661457

  16. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.

    PubMed

    Stoddart, Leigh A; White, Carl W; Nguyen, Kim; Hill, Stephen J; Pfleger, Kevin D G

    2016-10-01

    Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd ) and competition (Ki ) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super-bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc. © 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on

  17. An efficient reconstruction method for bioluminescence tomography based on two-step iterative shrinkage approach

    NASA Astrophysics Data System (ADS)

    Guo, Wei; Jia, Kebin; Tian, Jie; Han, Dong; Liu, Xueyan; Wu, Ping; Feng, Jinchao; Yang, Xin

    2012-03-01

    Among many molecular imaging modalities, Bioluminescence tomography (BLT) is an important optical molecular imaging modality. Due to its unique advantages in specificity, sensitivity, cost-effectiveness and low background noise, BLT is widely studied for live small animal imaging. Since only the photon distribution over the surface is measurable and the photo propagation with biological tissue is highly diffusive, BLT is often an ill-posed problem and may bear multiple solutions and aberrant reconstruction in the presence of measurement noise and optical parameter mismatches. For many BLT practical applications, such as early detection of tumors, the volumes of the light sources are very small compared with the whole body. Therefore, the L1-norm sparsity regularization has been used to take advantage of the sparsity prior knowledge and alleviate the ill-posedness of the problem. Iterative shrinkage (IST) algorithm is an important research achievement in a field of compressed sensing and widely applied in sparse signal reconstruction. However, the convergence rate of IST algorithm depends heavily on the linear operator. When the problem is ill-posed, it becomes very slow. In this paper, we present a sparsity regularization reconstruction method for BLT based on the two-step iterated shrinkage approach. By employing Two-step strategy of iterative reweighted shrinkage (IRS) to improve IST, the proposed method shows faster convergence rate and better adaptability for BLT. The simulation experiments with mouse atlas were conducted to evaluate the performance of proposed method. By contrast, the proposed method can obtain the stable and comparable reconstruction solution with less number of iterations.

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

    PubMed

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

    2014-12-01

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

  19. Bioluminescent Indicator for Highly Sensitive Analysis of Estrogenic Activity in a Cell-Based Format.

    PubMed

    Takenouchi, Osamu; Kanno, Akira; Takakura, Hideo; Hattori, Mitsuru; Ozawa, Takeaki

    2016-11-16

    Estrogens regulate different physiological systems with wide ranges of concentrations. The rapid analysis of estrogens is crucially important for drug discovery and medical diagnosis, but quantitation of nanomolar estrogens in live cells persists as an important challenge. We herein describe a bioluminescent indicator used to detect low concentrations of estrogens quantitatively with a high signal-to-background ratio. The indicator comprises a ligand-binding domain of an estrogen receptor connected with its binding peptide, which is sandwiched between split fragments of a luciferase mutant. Results show that the indicator recovered its bioluminescence upon binding to 17β-estradiol at concentrations higher than 1.0 × 10(-10) M. The indicator was reactive to agonists but did not respond to antagonists. The indicator is expected to be applicable for rapid screening estrogenic compounds and inhibitors, facilitating the discovery of drug candidates in a high-throughput manner.

  20. Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening

    PubMed Central

    Leippe, Donna; Sobol, Mary; Vidugiris, Gediminas; Zhou, Wenhui; Meisenheimer, Poncho; Gautam, Prson; Wennerberg, Krister; Cali, James J.

    2014-01-01

    Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z′ value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels. PMID:25506801

  1. Development and Application of a Bioluminescence-Based Test for Assimilable Organic Carbon in Reclaimed Waters▿

    PubMed Central

    Weinrich, Lauren A.; Giraldo, Eugenio; LeChevallier, Mark W.

    2009-01-01

    Assimilable organic carbon (AOC) is an important parameter governing the growth of heterotrophic bacteria in drinking water. Despite the recognition that variations in treatment practices (e.g., disinfection, coagulation, selection of filter media, and watershed protection) can have dramatic impacts on AOC levels in drinking water, few water utilities routinely measure AOC levels because of the difficulty of the method. To simplify the method, the Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX test bacteria were mutagenized by using luxCDABE operon fusion and inducible transposons to produce bioluminescent strains. The growth of these strains can easily be monitored with a programmable luminometer to determine the maximum cell yield via luminescence readings, and these values can be fitted to the classical Monod growth curve to determine bacterial growth kinetics and the maximum growth rate. Standard curves using acetate carbon (at concentrations ranging from 0 to 1,000 μg/liter) resulted in coefficients of determination (r2) between luminescence units and acetate carbon levels of 0.95 for P-17 and 0.89 for NOX. The bioluminescence test was used to monitor reclaimed water, in which average AOC levels range between 150 and 1,400 μg/liter acetate carbon equivalents. Comparison of the conventional AOC assay and the bioluminescent assay produced an r2 of 0.92. PMID:19820156

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

    NASA Astrophysics Data System (ADS)

    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.

  3. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    PubMed

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  4. Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

    PubMed Central

    Cosette, Jeremie; Ben Abdelwahed, Rym; Donnou-Triffault, Sabrina; Sautès-Fridman, Catherine

    2016-01-01

    Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies. PMID:27501019

  5. Development of a Filtration-Based Bioluminescence Assay for Detection of Microorganisms in Tea Beverages.

    PubMed

    Shinozaki, Yohei; Igarashi, Toshinori; Harada, Yasuhiro

    2016-03-01

    The market for tea drinks as healthy beverages has been steadily expanding, and ready-to-drink beverages in polyethylene terephthalate bottles have been popular. To more rapidly and accurately test tea beverages bottled in polyethylene terephthalate for microbial contamination, a newly developed filtration device and a washing method with a commercial bioluminescence assay were combined to detect low numbers of bacterial spores, fungal conidia, and ascospores. Washing buffers were formulated with nonionic detergents from the Tween series. Commercially available tea beverages were used to evaluate the filtration capacity of the filtration device, the effect of washing buffers, and the performance of the assay. The assay was tested with serially diluted suspensions of colonies of two bacterial strains, spores of three Bacillus strains, conidia of five fungal strains, and ascospores of four fungal strains. The filtration device enabled filtration of a large sample volume (100 to 500 ml), and the washing buffer significantly decreased the background bioluminescence intensity of tea samples when compared with the no-washing method. Low numbers (1 to 10 CFU/100 ml) of the tested strains of bacteria were detected within 8 to 18 h of cultivation, and fungi were detected within 24 to 48 h. Furthermore, a whole bottle (500 ml) of mixed tea was filtered through the filtration device and microbes were detected. This method could be used for quality control of bottled beverages without preincubation.

  6. Reconstruction Method for In Vivo Bioluminescence Tomography Based on the Split Bregman Iterative and Surrogate Functions.

    PubMed

    Zhang, Shuang; Wang, Kun; Liu, Hongbo; Leng, Chengcai; Gao, Yuan; Tian, Jie

    2017-04-01

    Bioluminescence tomography (BLT) can provide in vivo three-dimensional (3D) images for quantitative analysis of biological processes in preclinical small animal studies, which is superior than the conventional planar bioluminescence imaging. However, to reconstruct light sources under the skin in 3D with desirable accuracy and efficiency, BLT has to face the ill-posed and ill-conditioned inverse problem. In this paper, we developed a new method for BLT reconstruction, which utilized the mathematical strategies of the split Bregman iterative and surrogate functions (SBISF) method. The proposed method considered the sparsity characteristic of the reconstructed sources. Thus, the sparsity itself was regarded as a kind of a priori information, and the sparse regularization is incorporated, which can accurately locate the position of the sources. Numerical simulation experiments of multisource cases with comparative analyses were performed to evaluate the performance of the proposed method. Then, a bead-implanted mouse and a breast cancer xenograft mouse model were employed to validate the feasibility of this method in in vivo experiments. The results of both simulation and in vivo experiments indicated that comparing with the L1-norm iteration shrinkage method and non-monotone spectral projected gradient pursuit method, the proposed SBISF method provided the smallest position error with the least amount of time consumption. The SBISF method is able to achieve high accuracy and high efficiency in BLT reconstruction and hold great potential for making BLT more practical in small animal studies.

  7. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology

    PubMed Central

    Thorne, Natasha; Inglese, James; Auld, Douglas S.

    2010-01-01

    Summary Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell-based assays these inhibitors can act in a counter-intuitive way –leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition/stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multi-substrate-adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here. PMID:20609414

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

    SciTech Connect

    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.

  9. Biological toxicity of cellulose nanocrystals (CNCs) against the luxCDABE-based bioluminescent bioreporter Escherichia coli 652T7.

    PubMed

    Du, Liyu; Arnholt, Kelly; Ripp, Steven; Sayler, Gary; Wang, Siqun; Liang, Chenghua; Wang, Jingkuan; Zhuang, Jie

    2015-12-01

    The aim of this study was to evaluate the biological toxicity of cellulose nanocrystals (CNCs) using the constitutively bioluminescent luxCDABE-based bioreporter Escherichia coli 652T7. The effects of CNCs on E. c oli 652T7 biotoxicity were investigated at different CNC concentrations, reaction times, and IC50 values. CNC toxicity was also compared with and without ultrasonic dispersion to establish dispersibility effects. The results demonstrated that CNCs were not significantly toxic at concentrations at or below 250 mg/L. At concentrations higher than 300 mg/L, toxicity increased linearly as CNC concentrations increased up to 2000 mg/L. IC50 calculations demonstrated an increase in cytotoxicity as CNC exposure times increased, and elevated dispersibility of the CNCs were shown to increase cytotoxicity effects. These results suggest that CNCs can impact microbial populations if elevated concentration thresholds are met.

  10. Bioluminescence-Based High-Throughput Screen Identifies Pharmacological Agents That Target Neurotransmitter Signaling in Small Cell Lung Carcinoma

    PubMed Central

    Improgo, Ma. Reina D.; Johnson, Christopher W.; Tapper, Andrew R.; Gardner, Paul D.

    2011-01-01

    Background Frontline treatment of small cell lung carcinoma (SCLC) relies heavily on chemotherapeutic agents and radiation therapy. Though SCLC patients respond well to initial cycles of chemotherapy, they eventually develop resistance. Identification of novel therapies against SCLC is therefore imperative. Methods and Findings We have designed a bioluminescence-based cell viability assay for high-throughput screening of anti-SCLC agents. The assay was first validated via standard pharmacological agents and RNA interference using two human SCLC cell lines. We then utilized the assay in a high-throughput screen using the LOPAC1280 compound library. The screening identified several drugs that target classic cancer signaling pathways as well as neuroendocrine markers in SCLC. In particular, perturbation of dopaminergic and serotonergic signaling inhibits SCLC cell viability. Conclusions The convergence of our pharmacological data with key SCLC pathway components reiterates the importance of neurotransmitter signaling in SCLC etiology and points to possible leads for drug development. PMID:21931655

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

    PubMed

    Song, Qinxin; Wei, Guijiang; Zhou, Guohua

    2014-07-01

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

  12. The Chemical Basis of Fungal Bioluminescence.

    PubMed

    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

    2015-07-06

    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.

  13. Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.

    PubMed

    Curtis, Allison; Calabro, Katherine; Galarneau, Jean-Rene; Bigio, Irving J; Krucker, Thomas

    2011-12-01

    Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57Bl/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly pigmented skin was attenuated an average of 90%. Positive correlation was found between reflectance measurements and bioluminescence signal loss. A correction scheme is proposed based on this correlation, but signal variation due to non-melanin scattering and absorption sources introduce significant errors. Advanced spectral reflectance analysis will be necessary to develop a more reliable correction method in the future. Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required.

  14. A Causal Relation between Bioluminescence and Oxygen to Quantify the Cell Niche

    PubMed Central

    Lambrechts, Dennis; Roeffaers, Maarten; Goossens, Karel; Hofkens, Johan; 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 cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner. PMID:24840204

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

    PubMed

    Kiddle, Guy; Hardinge, Patrick; Buttigieg, Neil; Gandelman, Olga; Pereira, Clint; McElgunn, Cathal J; Rizzoli, Manuela; Jackson, Rebecca; Appleton, Nigel; Moore, Cathy; Tisi, Laurence C; Murray, James A H

    2012-04-30

    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. 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. 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 within a reaction must be controlled to ensure

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

    PubMed Central

    2012-01-01

    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 within a reaction must

  17. Combining fluorescence and bioluminescence microscopy.

    PubMed

    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.

  18. Hybrid radiosity-SP{sub 3} equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    SciTech Connect

    Chen, Xueli E-mail: jimleung@mail.xidian.edu.cn; Zhang, Qitan; Yang, Defu; Liang, Jimin E-mail: jimleung@mail.xidian.edu.cn

    2014-01-14

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP{sub 3} equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP{sub 3}) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  19. Circadian control sheds light on fungal bioluminescence.

    PubMed

    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.

  20. Circadian Control Sheds Light on Fungal Bioluminescence

    PubMed Central

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

    2015-01-01

    Summary 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] 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 by-product 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 the luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millenia [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. PMID:25802150

  1. Engineering bioluminescent proteins: expanding their analytical potential.

    PubMed

    Rowe, Laura; Dikici, Emre; Daunert, Sylvia

    2009-11-01

    Bioluminescent proteins are used in a plethora of analytical methods, from ultrasensitive assay development to the in vivo imaging of cellular processes. This article reviews the most pertinent current bioluminescent-protein-based technologies and suggests the future direction of this vein of research. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

  2. Laser-induced bioluminescence

    SciTech Connect

    Hickman, G.D.; Lynch, R.V. III

    1981-01-01

    A project has been initiated to determine the feasibility of developing a complete airborne remote sensing system for rapidly mapping high concentration patches of bioluminescent organisms in the world's oceans. Conceptually, this system would be composed of a laser illuminator to induce bioluminescence and a low light level image intensifier for detection of light. Initial laboratory measurements consisted of using a 2-J flash lamp pulsed optical dye laser to excite bioluminescence in the marine dinoflagellate Pyrocustis lunula at ambient temperature using Rhodamine 6G as the lasing dye (585 nm) and a laser pulse width of 1 microsec. After a latency period of 15-20 msec, the bioluminescence maximum occurred in the blue (480 nm is the wavelength maximum for most dinoflagellate bioluminescence) with the peaking occurring approximately 65 msec after the laser pulse. Planned experiments will investigate the effect of different excitation wavelengths and energies at various temperatures and salinities of the cultures.

  3. Detection of Salmonella in Foods Using a Reference PN-ISO Method and an Alternative Method Based on Loop-mediated Isothermal Amplification Coupled with Bioluminescence.

    PubMed

    Sarowska, Jolanta; Frej-Mądrzak, Magdalena; Jama-Kmiecik, Agnieszka; Kilian, Anna; Teryks-Wołyniec, Dorota; Choroszy-Król, Irena

    2016-01-01

    Salmonella, one of the primary indicators of food safety, is a common cause of food poisoning of an epidemic nature around the world. These microorganisms can colonize the gastrointestinal tract of both people and animals, and next contaminate not only eggs, milk, meat and dairy products, but also vegetables, fruit, grains and even spices. The aim of this study was to analyze the frequency of detection of Salmonella spp. in food samples using a reference PN-ISO method and an alternative method based on loop-mediated isothermal amplification (LAMP) coupled with bioluminescence. Food samples were obtained in cooperation with the State Sanitary-Epidemiological Station in Wrocław. Dairy products, meat, fish, pastry and confectionery, vegetables, herbs and spices were analyzed. The food samples were examined using a standard culturing method according to PN-ISO 6579:2003 for Salmonella spp. and an alternative method based on the isothermal amplification and bioluminescence phenomenon using the 3M MDS device. In 399 tested food samples in 8 materials, using both the reference and the alternative LAMP-based method, the presence of salmonella was confirmed. The results obtained show the 100% sensitivity, specificity and accuracy of the presented alternative, LAMP-based technique compared to the reference PN-ISO method. The alternative method using isothermal amplification and bioluminescence makes it possible to detect Salmonella in foods in a much shorter time than the referential culturing method.

  4. Firefly luciferase-based dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis.

    PubMed

    Sun, Amy; Hou, Lewis; Prugpichailers, Tiffany; Dunkel, Jason; Kalani, Maziyar A; Chen, Xiaoyuan; Kalani, M Yashar S; Tse, Victor

    2010-04-01

    Bioluminescence imaging (BLI) is emerging as a cost-effective, high-throughput, noninvasive, and sensitive imaging modality to monitor cell growth and trafficking. We describe the use of dynamic BLI as a noninvasive method of assessing vessel permeability during brain tumor growth. With the use of stereotactic technique, 10 firefly luciferase-transfected GL26 mouse glioblastoma multiforme cells were injected into the brains of C57BL/6 mice (n = 80). After intraperitoneal injection of D-luciferin (150 mg/kg), serial dynamic BLI was performed at 1-minute intervals (30 seconds exposure) every 2 to 3 days until death of the animals. The maximum intensity was used as an indirect measurement of tumor growth. The adjusted slope of initial intensity (I90/Im) was used as a proxy to monitor the flow rate of blood into the vascular tree. Using a modified Evans blue perfusion protocol, we calculated the relative permeability of the vascular tree at various time points. Daily maximum intensity correlated strongly with tumor volume. At postinjection day 23, histology and BLI demonstrated an exponential growth of the tumor mass. Slopes were calculated to reflect the flow in the vessels feeding the tumor (adjusted slope = I90/Im). The increase in BLI intensity was correlated with a decrease in adjusted slope, reflecting a decrease in the rate of blood flow as tumor volume increased (y = 93.8e-0.49, R2 = 0.63). Examination of calculated slopes revealed a peak in permeability around postinjection day 20 (n = 42, P < .02 by 1-way analysis of variance) and showed a downward trend in relation to both postinjection day and maximum intensity observed; as angiogenesis progressed, tumor vessel caliber increased dramatically, resulting in sluggish but increased flow. This trend was correlated with Evans blue histology, revealing an increase in Evans blue dye uptake into the tumor, as slope calculated by BLI increases. Dynamic BLI is a practical, noninvasive technique that can

  5. Development and Application of a Cellular, Gain-of-Signal, Bioluminescent Reporter Screen for Inhibitors of Type II Secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei

    PubMed Central

    Moir, Donald T.; Di, Ming; Wong, Erica; Moore, Richard A.; Schweizer, Herbert P.; Woods, Donald E.; Bowlin, Terry L.

    2011-01-01

    The type II secretion (T2S) system in Gram-negative bacteria is comprised of the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, we used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive up-regulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the IPTG concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73,000) were detected as primary hits due to stimulation of luminescence with a z-score ≥5. Direct secretion assays of the 9 most potent hits, representing 5 chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm, but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 – 25 μM. In addition, 7 of the 9 compounds also inhibited the T2S-mediated extracellular secretion of phospholipases C by P. aeruginosa and of protease activity by Burkholderia pseudomallei. PMID:21602485

  6. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  7. NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence

    PubMed Central

    Cai, Weibo

    2016-01-01

    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 non-ideal 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 non-biased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

  8. Bioluminescence in the Sea

    NASA Astrophysics Data System (ADS)

    Haddock, Steven H. D.; Moline, Mark A.; Case, James F.

    2010-01-01

    Bioluminescence spans all oceanic dimensions and has evolved many times—from bacteria to fish—to powerfully influence behavioral and ecosystem dynamics. New methods and technology have brought great advances in understanding of the molecular basis of bioluminescence, its physiological control, and its significance in marine communities. Novel tools derived from understanding the chemistry of natural light-producing molecules have led to countless valuable applications, culminating recently in a related Nobel Prize. Marine organisms utilize bioluminescence for vital functions ranging from defense to reproduction. To understand these interactions and the distributions of luminous organisms, new instruments and platforms allow observations on individual to oceanographic scales. This review explores recent advances, including the chemical and molecular, phylogenetic and functional, community and oceanographic aspects of bioluminescence.

  9. A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

    PubMed Central

    Shivak, Dylan J.; MacKenzie, Keith D.; Watson, Nikole L.; Pasternak, J. Alex; Jones, Brian D.; Wang, Yejun; DeVinney, Rebekah; Wilson, Heather L.; Surette, Michael G.

    2016-01-01

    ABSTRACT Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ70-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10−8 to 10−10 in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCE This article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the

  10. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    PubMed

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  11. A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos.

    PubMed

    Teranishi, Katsunori

    2017-01-05

    The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold-water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra-weak, for living gills and luminescence activation for non-bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid, which were identified as in vivo bioluminescence-activating components. Original bioluminescence and bioluminescence produced from the addition of trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN3 , whereas the luminescence produced form the combination of NADPH and hispidin in thawed non-bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN3 . Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.

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

    PubMed

    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.

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

    PubMed Central

    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

  14. In vitro influence of hypoxia on bioluminescence imaging in brain tumor cells

    NASA Astrophysics Data System (ADS)

    Moriyama, Eduardo H.; Jarvi, Mark; Niedre, Mark; Mocanu, Joseph D.; Moriyama, Yumi; Li, Buhong; Lilge, Lothar; Wilson, Brian C.

    2007-02-01

    Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9L luc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO II levels below about 5%, falling by approximately 50% at 0.2% pO II. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.

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

    NASA Astrophysics Data System (ADS)

    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. Detection of nitrate/nitrite bioavailability in wastewater using a luxCDABE-based Klebsiella oxytoca bioluminescent bioreporter.

    PubMed

    Abd-El-Haleem, Desouky; Ripp, Steven; Zaki, Sahar; Sayler, Gary S

    2007-08-01

    In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate (R2 = 0.98), 1 to 6 ppm for nitrite (R2 = 0.99), and 1 to 7 ppm for ammonium (R2 = 0.99). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.

  17. Image-guided simulation for bioluminescence tomographic imaging

    NASA Astrophysics Data System (ADS)

    Kumar, Durairaj; Cong, Wenxiang; Thiesse, Jacqueline; Nixon, Earl; Meinel, John, Jr.; Cong, Alex; McLennan, Geoffrey; Hoffman, Eric A.; Ming, Jiang; Wang, Ge

    2005-04-01

    Noninvasive imaging of the reporter gene expression based on bioluminescence is playing an important role in the areas of cancer biology, cell biology, and gene therapy. The central problem for the bioluminescence tomography (BLT) we are developing is to reconstruct the underlying bioluminescent source distribution in a small animal using a modality fusion approach. To solve this inversion problem, a mathematical model of the mouse is built from a CT/micro-CT scan, which enables the assignment of optical parameters to various regions in the model. This optical geometrical model is used in the Monte Carlo simulation to calculate the flux distribution on the animal body surface, as a key part of the BLT process. The model development necessitates approximations in surface simplification, and so on. It leads to the model mismatches of different kinds. To overcome such discrepancies, instead of developing a mathematical model, segmented CT images are directly used in our simulation software. While the simulation code is executed, those images that are relevant are assessed according to the location of the propagating photon. Depending upon the segmentation rules including the pixel value range, appropriate optical parameters are selected for statistical sampling of the free path and weight of the photon. In this paper, we report luminescence experiments using a physical mouse phantom to evaluate this image-guided simulation procedure, which suggest both the feasibility and some advantages of this technique over the existing methods.

  18. Monitoring of environmental pollutants by bioluminescent bacteria.

    PubMed

    Girotti, Stefano; Ferri, Elida Nora; Fumo, Maria Grazia; Maiolini, Elisabetta

    2008-02-04

    This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000-2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

  19. Metal-enhanced bioluminescence: An approach for monitoring biological luminescent processes

    NASA Astrophysics Data System (ADS)

    Eltzov, Evgeni; Prilutsky, Daria; Kushmaro, Ariel; Marks, Robert S.; Geddes, Chris D.

    2009-02-01

    In this letter, the observation of metal (plasmon)-enhanced bioluminescence is reported. Bacteria, which are capable of generating specific bioluminescence signatures upon metabolic changes (general toxicity), have been studied from both glass and silvered glass microwell bottoms, where the silvered microwells have been modified with surface deposited silver island films (SiFs). The presence of the SiFs plasmon amplifies the near-field bioluminescence signatures, ≈<50 nm from the surface, enabling amplified detection of the reporter bioluminescence indicating sample toxicity. Using our approach a greater than fivefold enhancement in far-field bioluminescence occurs with much greater enhancements in the near-field predicted.

  20. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  1. Effect of heavy atoms in bioluminescent reactions.

    PubMed

    Kirillova, Tamara N; Kudryasheva, Nadezhda S

    2007-03-01

    Bioluminescent reactions of luminous organisms are excellent models for studying the effects of heavy atoms on enzymatic processes. The effects of potassium halides with halide anions of different atomic weight were compared in bioluminescent reactions of the firefly (Luciola mingrelica), a marine coelenterate (Obelia longissima), and a marine bacterium (Photobacterium leiognathi). Two mechanisms of the effects of the halides were examined-the physicochemical effect of the external heavy atom, based on spin-orbit interactions in electron-excited structures, and the biochemical effect, i.e. interactions with the enzymes resulting in changes of enzymatic activity. The physicochemical effect was evaluated by using photoexcitation of model fluorescent compounds (flavin mononucleotide, firefly luciferin, and coelenteramide) of similar structure to the bioluminescence emitters. The bioluminescent and photoluminescent inhibition coefficients were calculated and compared for the luminous organisms to evaluate the relative contributions of the two mechanisms. The biochemical mechanism was found to be dominant. Hence, the bioluminescent reactions can be used as assays to monitor enzyme inhibition, in metabolic processes, by Br or I-containing compounds.

  2. Immobilized Bioluminescent Reagents in Flow Injection Analysis.

    NASA Astrophysics Data System (ADS)

    Nabi, Abdul

    Available from UMI in association with The British Library. Bioluminescent reactions exhibits two important characteristics from an analytical viewpoint; they are selective and highly sensitive. Furthermore, bioluminescent emissions are easily measured with a simple flow-through detector based on a photomultiplier tube and the rapid and reproducible mixing of sample and expensive reagent is best achieved by a flow injection manifold. The two most important bioluminescent systems are the enzyme (luciferase)/substrate (luciferin) combinations extracted from fireflies (Photinus pyralis) and marine bacteria (Virio harveyi) which requires ATP and NAD(P)H respectively as cofactors. Reactions that generate or consume these cofactors can also be coupled to the bioluminescent reaction to provide assays for a wide range of clinically important species. A flow injection manifold for the study of bioluminescent reactions is described, as are procedures for the extraction, purification and immobilization of firefly and bacterial luciferase and oxidoreductase. Results are presented for the determination of ATP using firefly system and the determination of other enzymes and substrates participating in ATP-converting reactions e.g. creatine kinase, ATP-sulphurylase, pyruvate kinase, creatine phosphate, pyrophosphate and phophoenolypyruvate. Similarly results are presented for the determination of NAD(P)H, FMN, FMNH_2 and several dehydrogenases which produce NAD(P)H and their substrates, e.g. alcohol, L-lactate, L-malate, L-glutamate, Glucose-6-phosphate and primary bile acid.

  3. Thoughts on the diversity of convergent evolution of bioluminescence on earth

    NASA Astrophysics Data System (ADS)

    Waldenmaier, Hans E.; Oliveira, Anderson G.; Stevani, Cassius V.

    2012-10-01

    The widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms. Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms. Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

  4. Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

    PubMed Central

    Haddix, Pryce L.; Shaw, Nancy J.; LeChevallier, Mark W.

    2004-01-01

    The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection. PMID:14766564

  5. Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria.

    PubMed

    Haddix, Pryce L; Shaw, Nancy J; LeChevallier, Mark W

    2004-02-01

    The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.

  6. Efficiency of peracetic acid in inactivating bacteria, viruses, and spores in water determined with ATP bioluminescence, quantitative PCR, and culture-based methods.

    PubMed

    Park, Eunyoung; Lee, Cheonghoon; Bisesi, Michael; Lee, Jiyoung

    2014-03-01

    The disinfection efficiency of peracetic acid (PAA) was investigated on three microbial types using three different methods (filtration-based ATP (adenosine-triphosphate) bioluminescence, quantitative polymerase chain reaction (qPCR), culture-based method). Fecal indicator bacteria (Enterococcus faecium), virus indicator (male-specific (F(+)) coliphages (coliphages)), and protozoa disinfection surrogate (Bacillus subtilis spores (spores)) were tested. The mode of action for spore disinfection was visualized using scanning electron microscopy. The results indicated that PAA concentrations of 5 ppm (contact time: 5 min), 50 ppm (10 min), and 3,000 ppm (5 min) were needed to achieve 3-log reduction of E. faecium, coliphages, and spores, respectively. Scanning electron microscopy observation showed that PAA targets the external layers of spores. The lower reduction rates of tested microbes measured with qPCR suggest that qPCR may overestimate the surviving microbes. Collectively, PAA showed broad disinfection efficiency (susceptibility: E. faecium > coliphages > spores). For E. faecium and spores, ATP bioluminescence was substantially faster (∼5 min) than culture-based method (>24 h) and qPCR (2-3 h). This study suggests PAA as an effective alternative to inactivate broad types of microbial contaminants in water. Together with the use of rapid detection methods, this approach can be useful for urgent situations when timely response is needed for ensuring water quality.

  7. Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase.

    PubMed

    Marques, Simone M; Peralta, Filipe; Esteves da Silva, Joaquim C G

    2009-02-15

    Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (D-LH(2)) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A-incubation time (t(inc)=15 min), inactivation time of the enzyme (t(inac)=2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP]=40 microM) and luciferase ([Luc]=0.1 microM); method B-concentrations of L-AMP ([L-AMP]=2 microM), luciferase ([Luc]=50 nM) and luciferin ([LH(2)]=30 microM). Method A has a linear response over the range of 0.1-20 microM of PPi, with a limit of detection (LOD) of 0.5 microM and a limit of quantitation (LOQ) of 1.8 microM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 microM PPi and 5.9% at 8 microM PPi. Method B has a linear response over the range of 0.75-6.0 microM of PPi, with LOD and LOQ of 0.624 and 2.23 microM, respectively, and a R.S.D. of 5.1% at 2.5 microM PPi and 4.9% at 5 microM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P(3)).

  8. Bioluminescence-based measurement of viability of Pseudomonas aeruginosa ATCC 9027 harbouring plasmid-based lux genes under the control of constitutive promoters.

    PubMed

    Shah, N; Naseby, D C

    2014-11-01

    Detection of microbial contamination in pharmaceuticals, food and cosmetics has been problematic for several decades. Numerous investigations highlight the urgency for novel methods; development of bioluminescent constructs allows real-time monitoring, rapid analysis and high-throughput screening of products. Microbial growth can be studied by measuring constitutive gene expression. The aim is to develop whole-cell microbial biosensors with Pseudomonas aeruginosa and quantify their growth rate by measuring constitutive expression of lux. Pseudomonas aeruginosa cells were genetically modified to produce bioluminescence constitutively. Strains were characterized by assessing their growth kinetics, plasmid stability and gene expression with bacterial replication. Furthermore, cell viability was measured by fluorescence quantification. Promoter strengths were evaluated by comparing bioluminescence (RLU) per colony-forming units (CFU) at various growth stages and related to promoter sequences. Promoter strength decreased in the order of P(lpp) > P(tat) > P(lysS) > P(ldcC) > P(spc) during exponential phase whilst P(tat) was stronger than P(lpp) during stationary phase. Good correlations between RLU and CFU at 24 h indicated a strong relationship for all bioluminescent strains; however, weaker correlations between RLU and CFU and between fluorescence (RFU) and CFU beyond 24 h indicated that a proportion of cells had lost the ability to culture. Equivalence analysis showed no significant difference between bioluminescence and plate counting for all five bioluminescent strains. Pseudomonas aeruginosa-containing P(tat) had steady bioluminescence when correlated to CFU (R > 0·9), and together with fluorescence data, it can be concluded that Ps. aeruginosa ATCC 9027 tat-pMElux is preferred for testing microbial viability. These whole-cell bioluminescent strains provide a platform for utilization in monitoring toxicity and contamination of compounds in environmental biology

  9. Bioluminescent bioreporter integrated circuit

    DOEpatents

    Simpson, Michael L.; Sayler, Gary S.; Paulus, Michael J.

    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.

  10. A Prototype Bioluminescence Photometer

    DTIC Science & Technology

    1982-02-01

    Seliger et al., 1962) provided maximum mechanical stimulation to dino - flagellates , and incorporated light baffles to permit continuous measurement...was deployed in a shallow coastal area relatively free of any mixing. The principle bioluminescent plankton were the dino - flagellates , as evidenced

  11. Synthetic Routes to Coelenterazine and Other Imidazo[1,2-a]pyrazin-3-one Luciferins: Essential Tools for Bioluminescence-Based Investigations.

    PubMed

    Coutant, Eloi P; Janin, Yves L

    2015-11-23

    In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2-a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence ("glow" systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2-a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Bioluminescence-based identification of nisin producers - a rapid and simple screening method for nisinogenic bacteria in food samples.

    PubMed

    Virolainen, Nina; Guglielmetti, Simone; Arioli, Stefania; Karp, Matti

    2012-08-17

    We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982-7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3 × 10(2)CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Bioluminescence Truth Data Measurement and Signature Detection

    DTIC Science & Technology

    2007-09-30

    concentration measurement algorithms, which permit identification to taxon (e.g. dinoflagellates or ctenophores ) based on flash kinetics...RELATED PROJECTS A closely related project entitled, “Monitoring bloom dynamics of a common coastal bioluminescent ctenophore ,” is being funded under

  14. Bioluminescence Truth Data Measurement and Signature Detection

    DTIC Science & Technology

    2006-01-01

    measurement algorithms, which permit identification to taxon (e.g. dinoflagellates or ctenophores ) based on flash kinetics. Figure 1: A graphic...closely related project entitled, “Monitoring bloom dynamics of a common coastal bioluminescent ctenophore ,” is being funded under Office of Naval

  15. The first report of luminescent liver tissue in fishes: evolution and structure of bioluminescent organs in the deep-sea naked barracudinas (Aulopiformes: Lestidiidae).

    PubMed

    Ghedotti, Michael J; Barton, Ryan W; Simons, Andrew M; Davis, Matthew P

    2015-03-01

    Bioluminescent organs that provide ventral camouflage are common among fishes in the meso-bathypelagic zones of the deep sea. However, the anatomical structures that have been modified to produce light vary substantially among different groups of fishes. Although the anatomical structure and evolutionary derivation of some of these organs have been well studied, the light organs of the naked barracudinas have received little scientific attention. This study describes the anatomy and evolution of bioluminescent organs in the Lestidiidae (naked barracudinas) in the context of a new phylogeny of barracudinas and closely related alepisauroid fishes. Gross and histological examination of bioluminescent organs or homologous structures from preserved museum specimens indicate that the ventral light organ is derived from hepatopancreatic tissue and that the antorbital spot in Lestrolepis is, in fact, a second dermal light organ. In the context of the phylogeny generated from DNA-sequence data from eight gene fragments (7 nuclear and 1 mitochondrial), a complex liver with a narrow ventral strand running along the ventral midline evolves first in the Lestidiidae. The ventral hepatopancreatic tissue later evolves into a ventral bioluminescent organ in the ancestor of Lestidium and Lestrolepis with the lineage leading to the genus Lestrolepis evolving a dermal antorbital bioluminescent organ, likely for light-intensity matching. This is the first described hepatopancreatic bioluminescent organ in fishes.

  16. Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation

    PubMed Central

    Chen, Ian Y.; Greve, Joan M.; Gheysens, Olivier; Willmann, Jürgen K.; Rodriguez-Porcel, Martin; Chu, Pauline; Sheikh, Ahmad Y.; Faranesh, Anthony Z.; Paulmurugan, Ramasamy; Yang, Phillip C.; Wu, Joseph C.; Gambhir, Sanjiv S.

    2014-01-01

    Purpose In this study, we compared firefly luciferase (Fluc) reporter gene and super-paramagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively. Procedures Rats (n=31) underwent an intramyocardial injection of cardiomyoblasts (2×106) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 μg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis. Results BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (≥16 days) and persistent MR signal (≥42 days). Conclusions Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells. PMID:19034584

  17. Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

    PubMed Central

    Krishnamoorthy, Archana

    2015-01-01

    Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases. PMID:26162874

  18. Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae.

    PubMed

    Krishnamoorthy, Archana; Robertson, J Brian

    2015-09-01

    Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases.

  19. Bioluminescence lights the way to food safety

    NASA Astrophysics Data System (ADS)

    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.

  20. Multicolor Bioluminescence Obtained Using Firefly Luciferin.

    PubMed

    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®".

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

    PubMed Central

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

    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 ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids. PMID:21742869

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

    SciTech Connect

    Chauhan, Archana; Layton, Alice; Williams, Daniel W; Smart, Abby E.; Ripp, Steven Anthony; Karpinets, Tatiana V; Brown, Steven D; Sayler, Gary Steven

    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.

  3. Theoretical Study of Dinoflagellate Bioluminescence.

    PubMed

    Wang, Ming-Yu; Liu, Ya-Jun

    2017-03-01

    Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin-luciferase one. However, the excited-state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin-luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.

  4. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  5. Bioluminescent bioreporter sensing of foodborne toxins

    NASA Astrophysics Data System (ADS)

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

    2004-06-01

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

  6. Bubble stimulation efficiency of dinoflagellate bioluminescence.

    PubMed

    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. Copyright © 2015 John Wiley & Sons, Ltd.

  7. A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin.

    PubMed

    Colepicolo, P; Camarero, V C; Nicolas, M T; Bassot, J M; Karnovsky, M L; Hastings, J W

    1990-02-01

    Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.

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

    PubMed

    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.

  9. Selected Least Studied but not Forgotten Bioluminescent Systems.

    PubMed

    Oba, Yuichi; Stevani, Cassius V; Oliveira, Anderson G; Tsarkova, Aleksandra S; Chepurnykh, Tatiana V; Yampolsky, Ilia V

    2017-03-01

    Bioluminescence is a form of chemiluminescence generated by luminous organisms. Luminous taxa have currently been reported from about 800 genera and probably over 10 000 species in the world. On the other hand, their bioluminescent systems, including chemical structures of luciferins/chromophores and the genes encoding luciferases/photoproteins, have been elucidated from only a few taxonomic groups, for example beetles, bacteria, dinoflagellates, ostracods and some cnidarians. Research efforts to understand unknown bioluminescence systems are being conducted around the world, and recently, for example, novel luciferin structures of luminous enchytraeid potworms and fungi were identified by the authors. In this study, we review the current status and perspectives, in the context of postgenomic era, of most likely novel but less-revealed bioluminescence systems of ten selected organisms: earthworm, parchment tubeworm, fireworm, scaleworm, limpet, millipede, brittle star, acorn worms, tunicate and shark, which indeed are the next focus of our international collaboration.

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

    PubMed

    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.

  11. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2010-01-01

    naval nighttime operations because the flow field associated with their motion stimulates naturally occurring plankton . In the littoral, the primary...sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...bioluminescent “signatures” of some swimming fish are distinct enough to differentiate species; nocturnally foraging predators may use bioluminescent

  12. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2011-09-30

    naval nighttime operations because the flow field associated with their motion stimulates naturally occurring plankton . In the littoral, the primary...sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...bioluminescent signatures of some swimming fish are distinct enough to differentiate species; nocturnally foraging predators may use bioluminescent

  13. A simple bioluminescent method for measuring D-amino acid oxidase activity.

    PubMed

    Bailey, T Spencer; Donor, Micah T; Naughton, Sean P; Pluth, Michael D

    2015-03-28

    D-Amino acid oxidase (DAO) plays important roles in regulating D-amino acid neurotransmitters and was recently identified as a key enzyme integral to hydrogen sulfide production from D-Cys. We report here the development of a simple biocompatible, bioluminescent method for measuring DAO activity based on the highly selective condensation of D-Cys with 6-hydroxy-2-cyanobenzothiazole (CBT-OH) to form D-luciferin.

  14. A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

    PubMed Central

    Hay, Anthony G.; Rice, James F.; Applegate, Bruce M.; Bright, Nathan G.; Sayler, Gary S.

    2000-01-01

    A bioreporter was made containing a tfdRPDII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R2 = 0.9825) in the range of 2.0 μM to 1.1 × 102 μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 102 μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues. PMID:11010925

  15. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay.

    PubMed

    Paciello, Lucia; Falco, Francesco Cristino; Landi, Carmine; Parascandola, Palma

    2013-03-05

    Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.

  16. A Multi-Camera System for Bioluminescence Tomography in Preclinical Oncology Research

    PubMed Central

    Lewis, Matthew A.; Richer, Edmond; Slavine, Nikolai V.; Kodibagkar, Vikram D.; Soesbe, Todd C.; Antich, Peter P.; Mason, Ralph P.

    2013-01-01

    Bioluminescent imaging (BLI) of cells expressing luciferase is a valuable noninvasive technique for investigating molecular events and tumor dynamics in the living animal. Current usage is often limited to planar imaging, but tomographic imaging can enhance the usefulness of this technique in quantitative biomedical studies by allowing accurate determination of tumor size and attribution of the emitted light to a specific organ or tissue. Bioluminescence tomography based on a single camera with source rotation or mirrors to provide additional views has previously been reported. We report here in vivo studies using a novel approach with multiple rotating cameras that, when combined with image reconstruction software, provides the desired representation of point source metastases and other small lesions. Comparison with MRI validated the ability to detect lung tumor colonization in mouse lung. PMID:26824926

  17. Mechanism and color modulation of fungal bioluminescence

    PubMed Central

    Kaskova, Zinaida M.; Dörr, Felipe A.; Petushkov, Valentin N.; Purtov, Konstantin V.; Tsarkova, Aleksandra S.; Rodionova, Natalja S.; Mineev, Konstantin S.; Guglya, Elena B.; Kotlobay, Alexey; Baleeva, Nadezhda S.; Baranov, Mikhail S.; Arseniev, Alexander S.; Gitelson, Josef I.; Lukyanov, Sergey; Suzuki, Yoshiki; Kanie, Shusei; Pinto, Ernani; Di Mascio, Paolo; Waldenmaier, Hans E.; Pereira, Tatiana A.; Carvalho, Rodrigo P.; Oliveira, Anderson G.; Oba, Yuichi; Bastos, Erick L.; Stevani, Cassius V.; Yampolsky, Ilia V.

    2017-01-01

    Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates. PMID:28508049

  18. Bioluminescence imaging: a shining future for cardiac regeneration.

    PubMed

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

    2013-06-01

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

  19. Bioluminescence imaging: a shining future for cardiac regeneration

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    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.

  1. Cloning and characterization of new bioluminescent proteins

    NASA Astrophysics Data System (ADS)

    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. Chemiluminescence and bioluminescence microbe detection

    NASA Technical Reports Server (NTRS)

    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.

  3. Chemiluminescence and bioluminescence microbe detection

    NASA Technical Reports Server (NTRS)

    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.

  4. Marine Bioluminescence: Mechanisms and Evaluation

    DTIC Science & Technology

    1999-09-30

    of the adaptive significance of bioluminescence. APPROACH Luminescence excitation mechanisms – Using the dinoflagellate Pyrocystis fusiformis as a...numbers in Pyrocystis noctiluca (autotroph) and Protoperidinium pellucidum (heterotroph), two major elements of luminescent phytoplankton in the

  5. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait.

    PubMed

    Martini, Séverine; Haddock, Steven H D

    2017-04-04

    The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea.

  6. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

    PubMed Central

    Martini, Séverine; Haddock, Steven H. D.

    2017-01-01

    The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea. PMID:28374789

  7. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

    NASA Astrophysics Data System (ADS)

    Martini, Séverine; Haddock, Steven H. D.

    2017-04-01

    The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea.

  8. Bioluminescence Potential Modeling and Forecasting

    DTIC Science & Technology

    2013-05-22

    bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from simple agitation of the water with one’s hand...history of brilliant displays of bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from...during the earlier stages of upwelling development. Later, the observed deep offshore BL potential maximum disappeared and became a shallower and much

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

    NASA Astrophysics Data System (ADS)

    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. On the pH dependent behavior of the firefly bioluminescence: protein dynamics and water content in the active pocket.

    PubMed

    Kim, Hyun Woo; Rhee, Young Min

    2013-06-20

    Understanding bioluminescence presents fascinating challenges for fundamental sciences and numerous opportunities for practical applications. As a representative example, the firefly bioluminescent system has been intensively studied in both experimental and computational areas. However, there are still remaining questions regarding especially the detailed protein dynamics and the mechanisms of its color modulation. Here, we report on the pH dependent behavior of the firefly bioluminescence primarily based on molecular dynamics simulations. We find that the overall protein structure is generally resilient to pH variations. As the protein does not exhibit any structural distortions that can affect the emission property, we next focus on the dynamics in the active pocket and its effect on color modulation by adopting different protonation states in the pocket. With this, we observe red-shifted emissions at acidic conditions as consistent with previous studies. Most importantly, we find that a water molecule in the active pocket can mediate flexible motions of neighboring groups, which can subsequently modify the emission properties to a substantial degree. Based on the observations, we propose that the active pocket is in a dry condition during the luminescence process. Our results highlight the importance of understanding the role of the dynamics near the active pocket in modulating bioluminescence.

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

    PubMed

    Tiernan, Aubrey R; Sambanis, Athanassios

    2017-02-01

    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.

  12. Enhanced Landweber algorithm via Bregman iterations for bioluminescence tomography

    NASA Astrophysics Data System (ADS)

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

    PubMed Central

    Nackerdien, Zeena E.; 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 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. PMID:18301749

  14. Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

    PubMed

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  15. Cumulative bioluminescence; A potential rapid test of drilling fluid toxicity: development study

    SciTech Connect

    Stiffey, A.V. )

    1992-03-01

    A new rapid test of drilling fluid toxicity is based on the spontaneous bioluminescence of Pyrocystis lunula, an easy-to-culture alga that vigorously responds to shear stress (mixing) by emitting a sharp burst of light. In contrast to other bioluminescence methods, a cumulative flux of light is measured with a photomultiplier that eliminates the effect of exposure time on test results. Light quenching, caused by the presence of a toxicant, results in the dose/response relationship (DSR) typical for the enzymatic reaction kinetics. The Michaelis-Menten (dissociation) constant is used as a direct measure of toxicity. The evaluation study involved multiple experiments with 60 samples of drilling fluids from the U.S. gulf coast, as well as such typical toxicants as diesel oil, mineral oil, and chrome lignosulfonate (CLS). In this paper, the results of the test error analysis and comparisons with the Microtox and Mysid shrimp assays are reported.

  16. Determination of pyruvic acid concentration using a bioluminescence system from Photobacterium leiognathi.

    PubMed

    Xuan, Guanhua; Lu, Xiaodong; Wang, Jingxue; Lin, Hong; Liu, Huihui

    2015-06-01

    A novel, highly sensitive and selective bacterial luminescence method for the detection of pyruvic acid (PA) is reported here. This method is based on a reaction system catalyzed by lactate dehydrogenase (LDH) with the bacterial luciferase-FMN:NADH oxidoreductase bioluminescence system in vitro. The reduced nicotinamide adenine dinucleotide (NADH) involved in the LDH reaction system could be quantitatively analyzed by the bioluminescence system. A good linear relationship between the luminescence intensity and pyruvic acid concentration was exhibited within the range of 0.00014-0.001 mol l(-1), and the pyruvic acid detection limit was found to be 8.537 × 10(-5) mol l(-1). This method was successfully applied to the detection of PA in quail serum with a good recovery of over 70%.

  17. A Photinus pyralis and Luciola italica chimeric firefly luciferase produces enhanced bioluminescence.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Davis, Audrey L; Behney, Curran E; Murtiashaw, Martha H

    2014-10-14

    We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.

  18. Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual-color bioluminescence reporter.

    PubMed

    Kwon, Hyuck Joon; Ohmiya, Yoshihiro; Yasuda, Kazunori

    2014-12-01

    A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red-emitting Phrixothrix hirtus luciferase (PxRe) and a blue-emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non-mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual-color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes.

  19. Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

    PubMed Central

    Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

    2017-01-01

    Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

  20. Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

    PubMed Central

    Sanseverino, John; Gupta, Rakesh K.; Layton, Alice C.; Patterson, Stacey S.; Ripp, Steven A.; Saidak, Leslie; Simpson, Michael L.; Schultz, T. Wayne; Sayler, Gary S.

    2005-01-01

    An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10−8 through 5.6 × 10−12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10−10 and (2.4 ± 1.0) × 10−10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10−11 to 2.8 × 10−9 M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment. PMID:16085836

  1. Using bioluminescent biosensors for hazard analysis and critical control point (HACCP) in wastewater control.

    PubMed

    Valat, C; Champiat, D; Degorce-Dumas, J R; Thomas, O

    2004-01-01

    Starting from a new approach for water pollution control and wastewater treatment plant management, the hazard analysis and critical control point (HACCP) quality concept, the interest for the development of new rapid and sensitive methods such as bioluminescence-based methods is evident. After an introduction of the HACCP procedure, a bibliographic study of the bioluminescence potentiality is presented and discussed.

  2. Vision and Bioluminescence in the Deep-sea Benthos

    NASA Astrophysics Data System (ADS)

    Frank, T. M.; Johnsen, S.; Bracken-Grissom, H.; Messing, C. G.; Widder, E.

    2016-02-01

    During a NOAA-OER funded research cruise, novel collecting techniques were used to collect live, deep-sea benthic animals for studies of bioluminescence and vision. True color images and emission spectra of bioluminescence were obtained from a number of species, including the spiral octocoral Iridogorgia sp., the sea fan Chrysogorgia sp., the sea pen Umbellula sp., and the caridean shrimp Heterocarpus oryx. Electrophysiological studies were conducted on 3 species of decapod crustaceans collected with methods that limited light damage to their photoreceptors. The caridean shrimp, Bathypalaemonella, collected from 1920 m, was always found in association with the bioluminescent spiral octocoral Iridogorgia. While moribund at the surface, enough data were obtained from one specimen to show different waveforms in response to short and long wavelength light, indicative of two different classes of photoreceptor cells. The chirostylid crab, Uroptychus nitidus, found in association with the bioluminescent sea fan, Chrysogorgia sp., also appears to possess two visual pigments, and if further analysis of data supports this preliminary observation, will be the 4th species of deep-sea, non-bioluminescent crustaceans possessing two visual pigments found in association with bioluminescent cnidarians. These four species also share another characteristic - the presence of one or two very long claws, which the crab species are known to use to pick items (possibly plankton stuck in the mucus) off their cnidarian hosts. These data support the previously presented hypothesis (Frank et al. 2012), that these crustaceans may be utilizing their dual visual pigment systems to distinguish between prey and host, based on spectral differences between pelagic and benthic bioluminescence.

  3. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2009-01-01

    to flow stimulation of naturally occurring plankton. In the littoral, the primary sources of bioluminescence are dinoflagellates, common unicellular ...littoral, the primary sources of bioluminescence are dinoflagellates, common unicellular plankton that exhibit a wide range of abundance. Dinoflagellate

  4. Integrating printed microfluidics with silicon photomultipliers for miniaturised and highly sensitive ATP bioluminescence detection.

    PubMed

    Santangelo, M F; Libertino, S; Turner, A P F; Filippini, D; Mak, W C

    2018-01-15

    Bioluminescence has been widely used for important biosensing applications such as the measurement of adenosine triphosphate (ATP), the energy unit in biological systems and an indicator of vital processes. The current technology for detection is mainly based on large equipment such as readers and imaging systems, which require intensive and time-consuming procedures. A miniaturised bioluminescence sensing system, which would allow sensitive and continuous monitoring of ATP, with an integrated and low-cost disposable microfluidic chamber for handling of biological samples, is highly desirable. Here, we report the design, fabrication and testing of 3D printed microfluidics chips coupled with silicon photomultipliers (SiPMs) for high sensitive real-time ATP detection. The 3D microfluidic chip reduces reactant consumption and facilitates solution delivery close to the SiPM to increase the detection efficiency. Our system detects ATP with a limit of detection (LoD) of 8nM and an analytical dynamic range between 15nM and 1µM, showing a stability error of 3%, and a reproducibility error below of 20%. We demonstrate the dynamic monitoring of ATP in a continuous-flow system exhibiting a fast response time, ~4s, and a full recovery to the baseline level within 17s. Moreover, the SiPM-based bioluminescence sensing system shows a similar analytical dynamic range for ATP detection to that of a full-size PerkinElmer laboratory luminescence reader. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Main Technological Advancements in Bacterial Bioluminescent Biosensors Over the Last Two Decades.

    PubMed

    Jouanneau, S; Durand, M J; Lahmar, A; Thouand, G

    2015-10-17

    Environmental quality assessment is an extensive field of research due to the permanent increase of the stringency imposed by the legislative framework. To complete the wide panel of measurement methods, essentially based on physicochemical tools, some scientists focused on the development of alternative biological methods such as those based on the use of bioluminescent bacteria biosensors. The first report dedicated to the development of such biosensors dates back to 1967 and describes an analytical system designed to address the problem of air toxicity assessment. Nevertheless the available technologies in the photosensitive sensors field were not mature enough and, as a result, limited biosensor development possibilities. For about 20 years, the wide democratisation of photosensors coupled with advances in the genetic engineering field have allowed the expansion of the scope of possibilities of bioluminescent bacterial biosensors, allowing a significant emergence of these biotechnologies. This chapter retraces the history of the main technological evolutions that bacterial bioluminescent biosensors have known over the last two decades. Graphical Abstract.

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

    PubMed

    Straube, Nicolas; Li, Chenhong; Claes, Julien M; Corrigan, Shannon; Naylor, Gavin J P

    2015-08-16

    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 level sampling of "squaloid" sharks and closely related taxa using aligned sequences derived from a targeted gene capture approach. The resulting phylogenetic estimate is further used to evaluate the age of first occurrence of bioluminescence in Squaliformes. Our dataset comprised 172 putative ortholog exon sequences. Phylogenetic estimates result in a fully resolved tree supporting a monophyletic lineage of Squaliformes excluding Echinorhinus. Non-luminous Squalidae are inferred to be the sister to a clade comprising all remaining Squaliform families. Our results suggest that the origin of photophores is coincident with an elevated diversification rate and the splitting of families Dalatiidae, Etmopteridae, Oxynotidae and Somniosidae at the transition of the Lower to the Upper Cretaceous. The presence of luminous organs was confirmed for the Sleeper shark genus Zameus. These results indicate that bioluminescence in sharks is not restricted solely to the families Etmopteridae and Dalatiidae as previously believed. The sister-clade to non-luminous Squalidae comprises five families. The presence of photophores is reported for extant members of three out of these five families based on results of this study, i.e. Lantern sharks (Etmopteridae), Kitefin sharks (Dalatiidae) and Sleeper sharks (Somniosidae). Our results suggest that the origin of luminous organs arose during the rapid diversification event that gave rise to the extant Squaliform families. These inferences are consistent with the

  7. Engineering Bioluminescent Proteins: Expanding their Analytical Potential

    PubMed Central

    Rowe, Laura; Dikici, Emre; Daunert, Sylvia

    2009-01-01

    Synopsis Bioluminescence has been observed in nature since the dawn of time, but now, scientists are harnessing it for analytical applications. Laura Rowe, Emre Dikici, and Sylvia Daunert of the University of Kentucky describe the origins of bioluminescent proteins and explore their uses in the modern chemistry laboratory. The cover features spectra of bioluminescent light superimposed on an image of jellyfish, which are a common source of bioluminescent proteins. Images courtesy of Emre Dikici and Shutterstock. PMID:19725502

  8. Bioluminescence imaging serves as a dynamic marker for guiding and assessing thermal treatment of cancer in a preclinical model.

    PubMed

    Au, Joyce T; Gonzalez, Lorena; Chen, Chun-Hao; Serganova, Inna; Fong, Yuman

    2012-09-01

    Bioluminescence has been harnessed as a dynamic imaging technique in research. This is a proof of principle study examining feasibility of using bioluminescent proteins as a marker to guide therapeutic ablation. Mesothelioma cancer cells (MSTO-Td) were transfected with a retroviral vector bearing firefly luciferase gene, plated in serial dilutions, and imaged to compare bioluminescence signal to cell number, determining threshold of bioluminescence detection. MSTO-Td cells were subjected to thermal treatment in a heated chamber; the bioluminescence signal and number of remaining live cancer cells were determined. Mice with MSTO-Td xenografts underwent electrocautery tumor ablation guided by bioluminescence imaging; bioluminescence signal and tumor size were monitored for 3 weeks. MSTO-Td cells emitted a bright, clear, bioluminescence signal that amplified with the cell number (P < .001) and was detectable with as few as 10 cells in cell culture. Bioluminescence decreased in a dose-dependent fashion upon thermal treatment as temperature increased from 37 to 70 °C (P < .001) and as treatment duration increased from 5 to 20 min (P < .001). This correlated with the number of remaining live MSTO-Td cells (Pearson coefficient = 0.865; P < .001). In mice, the bioluminescence signal correlated with tumor size posttreatment and effectively guided the ablation procedure to completion, achieving 0 % tumor recurrence. Bioluminescence imaging is a sensitive, real-time imaging approach; bioluminescence reporters such as firefly luciferase can assess and guide thermal treatment of cancer. This encourages research into bioluminescence imaging as a molecular technique with potential to target tumors via biomarkers and optimize thermal treatment procedures in a clinical setting.

  9. Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.

    PubMed

    Wiles, Siouxsie; Robertson, Brian D; Frankel, Gad; Kerton, Angela

    2009-01-01

    Bioluminescence is an excellent reporter system for analysing bacterial colonization and clearance dynamics in vivo. Many bacterial species have been rendered bioluminescent, allowing the sensitive detection of bacterial burden and metabolic activity in real-time and in situ in living animals. In this chapter we describe the protocols for characterizing in vivo infection models using bioluminescent bacteria: from real-time imaging in living animals by bioluminescence imaging (BLI) to ex vivo BLI of harvested organs and tissues and, finally, to quantification of bacterial numbers in organ and tissue homogenates by luminometry and viable counts. While the lux operon from Photorhabdus luminescens is ideally suited for use in such models, there may be times when alternative luciferases, such as those from the firefly (luc) or marine copepods (Gluc), may be more appropriate. Here we describe the protocols required to monitor colonization and clearance dynamics using bioluminescent bacteria that are lux-, luc-, or Gluc-positive.

  10. Spectrally resolved bioluminescence tomography using the reciprocity approach

    PubMed Central

    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 the measured data. Reconstructed images of bioluminescence are presented using both simulated complex and heterogeneous small animal models as well as real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence source using experimental data is obtained with 0.5 mm accuracy and it is shown that small (2.5 mm diameter) sources of up to 12.5 mm deep, within a complex mouse model, can be resolved accurately using a single view data collection strategy. Finally, using the reciprocity approach for image reconstruction, a dramatic improvement in computational time is shown without loss to image accuracy with both experimental and simulated data, potentially reducing computing time from 402 to 3.75 h. PMID:19070220

  11. Bioluminescent Mycena species from São Paulo, Brazil.

    PubMed

    Desjardin, Dennis E; Capelari, Marina; Stevani, Cassius

    2007-01-01

    Six species of bioluminescent agarics are described and illustrated from a single site in primary Atlantic Forest habitat in the Parque Estadual Turistico do Alto Ribeira, Sao Paulo State, Brazil. These include two new taxa of Mycena, viz. M. asterina and M. lucentipes. Luminescence in Mycena fera, M. singeri and M. discobasis is reported for the first time. In addition an undeterminable luminescent Mycena species is described and additional specimens of Gerronema viridilucens are documented. An accounting of known bioluminescent species of Mycena and a discussion of why they luminesce are presented.

  12. Bioluminescent Reaction by Immobilized Luciferase

    NASA Astrophysics Data System (ADS)

    Tanaka, Ryuta; Takahama, Eriko; Iinuma, Masataka; Ikeda, Takeshi; Kadoya, Yutaka; Kuroda, Akio

    We have investigated an effect of immobilization of luciferase molecules at the optical fiber end on a bioluminescent reaction. The time dependence of measured count rates of emitted photons has been analyzed by fitting with numerical solution of differential equations including the effect of the product-inhibitor and the deactivation of the luciferase. Through the analysis, we have successfully extracted kinetic constants such as, reaction rate, number of active luciferase molecules, etc. Ratio of active molecules to total luciferase molecules in immobilization was one order of magnitude lower than that in solution. The reaction rate of the bioluminescent process was also different from the one of free luciferase in solution.

  13. CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence.

    PubMed

    Viviani, V R; Okawachi, F M; Scorsato, V; Abdalla, F C

    2008-04-01

    The anatomic and biochemical origin of beetle bioluminescence is still poorly understood. Through CCD imaging we report that larvae and pupae of the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp emit a continuous weak glow throughout the entire body during all stages. This luminescence is especially developed after feeding, ecdysis and in the pupal stage, gradually disappearing as the cuticle becomes sclerotized and the adult emerges. This weak glow arises from the fat body, which consists of small lobes spread all over the body cavity. According to their pigmentation, these lobes can be divided in whitish and pinkish, and display different luciferase isozymes. Morphological studies suggest that the jelly-like ventral lanterns in the 8th abdominal segment evolved from these white lobes, providing a rationale for the widespread location of lanterns in larvae of different bioluminescent beetles. The biological and biochemical function of this weak diffuse bioluminescence is discussed in the context of the larval life-history.

  14. Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells

    NASA Astrophysics Data System (ADS)

    Liang, Yajie; Walczak, Piotr; Bulte, Jeff W. M.

    2012-01-01

    One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/- mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, red-shifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

  15. Bacterial bioluminescence as a lure for marine zooplankton and fish

    PubMed Central

    Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

    2012-01-01

    The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea. PMID:22203999

  16. Bacterial bioluminescence as a lure for marine zooplankton and fish.

    PubMed

    Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

    2012-01-17

    The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea.

  17. Iterative reconstruction for bioluminescence tomography with total variation regularization

    NASA Astrophysics Data System (ADS)

    Jin, Wenma; He, Yonghong

    2012-12-01

    Bioluminescence tomography(BLT) is an instrumental molecular imaging modality designed for the 3D location and quantification of bioluminescent sources distribution in vivo. In our context, the diffusion approximation(DA) to radiative transfer equation(RTE) is utilized to model the forward process of light propagation. Mathematically, the solution uniqueness does not hold for DA-based BLT which is an inverse source problem of partial differential equations and hence is highly ill-posed. In the current work, we concentrate on a general regularization framework for BLT with Bregman distance as data fidelity and total variation(TV) as regularization. Two specializations of the Bregman distance, the least squares(LS) distance and Kullback-Leibler(KL) divergence, which correspond to the Gaussian and Poisson environments respectively, are demonstrated and the resulting regularization problems are denoted as LS+TV and KL+TV. Based on the constrained Landweber(CL) scheme and expectation maximization(EM) algorithm for BLT, iterative algorithms for the LS+TV and KL+TV problems in the context of BLT are developed, which are denoted as CL-TV and EM-TV respectively. They are both essentially gradient-based algorithms alternatingly performing the standard CL or EM iteration step and the TV correction step which requires the solution of a weighted ROF model. Chambolle's duality-based approach is adapted and extended to solving the weighted ROF subproblem. Numerical experiments for a 3D heterogeneous mouse phantom are carried out and preliminary results are reported to verify and evaluate the proposed algorithms. It is found that for piecewise-constant sources both CL-TV and EM-TV outperform the conventional CL and EM algorithms for BLT.

  18. Light emission miracle in the sea and preeminent applications of bioluminescence in recent new biotechnology.

    PubMed

    Sharifian, Sana; Homaei, Ahmad; Hemmati, Roohullah; Khajeh, Khosro

    2017-07-01

    Bioluminescence is referred to the light emission by a living organism due to a specific biochemical reaction. This interesting feature of the organisms could highly influences behavioral and ecosystem dynamics. Luminescence, mostly observed in marine species, is generally higher in deep-living genera than in benthic or shallow organisms. However, among creatures living in land, fireflies, beetles, springtails and fungi have shown some bioluminescent activities. Classically, the emission of light is catalyzed by luciferase from a substrate. Interestingly, light-emitting organisms are more abundant and widespread in marine than terrestrial environments. Novel tools derived from understanding bioluminescent reactions have led to countless valuable applications in modern biotechnology and biochemical engineering. Here, we overview some main properties bioluminescence in marine organism from bacteria to fishes following the latest advances and new discoveries of state-of-the-art bioluminescent tools in molecular biology, bioluminescent bioassays and imaging. The overview showed available and wide biotechnological tools of bioluminescence take advantage of its high detectability, high sensitive, low toxic and quantum efficiency which make wide usage as reporter of many biological functions in different fields, such as studying bacterial pathogens, ecotoxicology, food toxicity, tracking cells of interest in vivo, protein-protein interactions, gene expression and circadian rhythms. With the recent invention of luminescent reporters, future possibilities for the development of additional reporter applications are promising. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Biofilm Ecology of Bioluminescent Bacteria

    DTIC Science & Technology

    1992-08-10

    F.V. Lamberti , Z. Policova, W. Zingg, C.J. van Oss, and A.W. Neumann. 1983. Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbiol. 46...of bacterial adhesion in a shear gradient with bioluminescence by a Pseudomonas fluorescens (lux) strain . J. Microbiol. Meth. 15:53-60. Mittelman, M.W

  20. Marine Bioluminescence: Mechanisms and Evaluation

    DTIC Science & Technology

    2000-09-30

    Quantification of filamentous actin was accomplished throughout the cell cycle. McDougall found that Pyrocystis . fusiformis cultures grown under...Bight study clearly shows a marked seasonality in per-cell bioluminescence as well as cell numbers in Pyrocystis noctiluca (autotroph) and

  1. Dynamic Modeling of Marine Bioluminescence and Night Time Leaving Radiance

    DTIC Science & Technology

    2012-09-30

    physical model is based on the Navy Coastal Ocean Model (NCOM), the biochemical model simulates dynamics of two sizes of phytoplankton, zooplankton ...layer of bioluminescent zooplankton were replaced by water masses advected from the northern coast of the bay with a relatively high presence of mostly

  2. Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

    PubMed Central

    Xu, Tingting; Ripp, Steven; Sayler, Gary S.; Close, Dan M.

    2014-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines

  3. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon.

    PubMed

    Yagur-Kroll, Sharon; Belkin, Shimshon

    2011-05-01

    Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction's substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

  4. Dynamic Camouflage in Benthic and Pelagic Cephalopods: An Interdisciplinary Approach to Crypsis Based on Color, Reflection, and Bioluminescence

    DTIC Science & Technology

    2009-01-01

    benthic octopus with base-layer iridophores, chromatophores and two rings of intense blue iridescence. Figure 2: The proposed Spatial...species. Left: Pterygioteuthis microlampas a) animal in white light b) counterillumination. Middle: Loligo opalescens. Right: Octopus bimaculoides...chromatophores that is our molecular team’s current model system for understanding reflectin self-assembly. 3. Octopus bimaculoides: a small, hardy

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

    SciTech Connect

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

  6. Leukocyte esterase-nitrite and bioluminescence assays as urine screens.

    PubMed Central

    Males, B M; Bartholomew, W R; Amsterdam, D

    1985-01-01

    The 1-min leukocyte esterase (LE)-nitrite test (Chemstrip 9; Biodynamics, Division of Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a bioluminescence assay (Monolight centrifugation method; Analytical Luminescence Laboratory, Inc., San Diego, Calif.) were tested for their efficacy as urine screens among 453 patients at a tertiary-care teaching hospital. Both methods had the capacity to exclude significant bacteriuria (greater than or equal to 10(5) CFU/ml) when compared with the results of conventional culture methods, with predictive values of 99 and 93%, respectively, for a negative test. Bioluminescence was the more accurate nonculture method used. Sensitivity and specificity values were 97 and 71%, respectively, for bioluminescence, 82 and 60%, respectively, for LE with nitrite, and 72 and 64%, respectively, for LE without nitrite. At reduced levels of bacteriuria less than 10(5) CFU/ml), the sensitivities of LE-nitrite and bioluminescence were decreased but comparable. The addition of protein and blood test results in the Chemstrip 9, along with LE-nitrite as bacteriuria indicators, were unsatisfactory because of the large numbers of false-positive results attributed to protein and blood determinations. LE activity as detected by the LE test was a poor predictor of significant bacteriuria in both male and female patients. The sensitivity (71%) and specificity (57%) of the LE test in male patients were significantly lower than those previously reported and varied with the patient population studied. PMID:3935662

  7. Metabolic self-organization of bioluminescent Escherichia coli.

    PubMed

    Simkus, Remigijus; Baronas, Romas

    2011-01-01

    A possible reason for the complexity of the signals produced by bioluminescent biosensors might be self-organization of the cells. In order to verify this possibility, bioluminescence images of cultures of lux gene reporter Escherichia coli were recorded for several hours after being placed into 8-10 mm diameter cylindrical containers. It was found that luminous cells distribute near the three-phase contact line, forming irregular azimuthal waves. As we show, space-time plots of quasi-one-dimensional bioluminescence measured along the contact line can be simulated by reaction-diffusion-chemotaxis equations, in which the reaction term for the cells is a logistic (autocatalytic) growth function. It was found that the growth rate of the luminous cells (~0.02 s(-1)) is >100 times higher than the growth rate of E. coli. We provide an explanation for this result by assuming that E. coli exhibits considerable respiratory flexibility (the ability of oxygen-induced switching from one metabolic pathway to another). According to the simple two-state model presented here, the number of oxic (luminous) cells grows at the expense of anoxic (dark) cells, whereas the total number of (oxic and anoxic) cells remains unchanged. It is conjectured that the corresponding reaction-diffusion-chemotaxis model for bioluminescence pattern formation can be considered as a model for the energy-taxis and metabolic self-organization in the population of the metabolically flexible bacteria under hypoxic conditions.

  8. Microtiter plate tests for segregation of bioluminescent bacteria.

    PubMed

    Šimkus, Remigijus; Meškienė, Rita; Ledas, Žilvinas; Baronas, Romas; Meškys, Rolandas

    2016-02-01

    It has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self-organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux-gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three-phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch-like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller-Segel-Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two-state) bacteria. The modified KSF model successfully simulated the observed switch-like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three-phase contact line region.

  9. Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor.

    PubMed

    Wimmer, T; Lorenz, B; Stieger, K

    2016-12-15

    Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca(2+) -regulated Photoproteins of Different Organisms.

    PubMed

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

    2017-03-01

    Upon binding their metal ion cofactors, Ca(2+) -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca(2+) -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca(2+) -regulated photoproteins.

  11. Near-infrared bioluminescent proteins for two-color multimodal imaging

    PubMed Central

    Rumyantsev, Konstantin A.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2016-01-01

    Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification. PMID:27833162

  12. Coelenterazine analogues emit red-shifted bioluminescence with NanoLuc.

    PubMed

    Shakhmin, Anton; Hall, Mary P; Machleidt, Thomas; Walker, Joel R; Wood, Keith V; Kirkland, Thomas A

    2017-10-03

    We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.

  13. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  14. Murine Bioluminescent Hepatic Tumour Model

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. PMID:20689502

  15. Uniqueness theorems in bioluminescence tomography.

    PubMed

    Wang, Ge; Li, Yi; Jiang, Ming

    2004-08-01

    Motivated by bioluminescent imaging needs for studies on gene therapy and other applications in the mouse models, a bioluminescence tomography (BLT) system is being developed in the University of Iowa. While the forward imaging model is described by the well-known diffusion equation, the inverse problem is to recover an internal bioluminescent source distribution subject to Cauchy data. Our primary goal in this paper is to establish the solution uniqueness for BLT under practical constraints despite the ill-posedness of the inverse problem in the general case. After a review on the inverse source literature, we demonstrate that in the general case the BLT solution is not unique by constructing the set of all the solutions to this inverse problem. Then, we show the uniqueness of the solution in the case of impulse sources. Finally, we present our main theorem that solid/hollow ball sources can be uniquely determined up to nonradiating sources. For better readability, the exact conditions for and rigorous proofs of the theorems are given in the Appendices. Further research directions are also discussed.

  16. Molecular-genetic imaging based on reporter gene expression.

    PubMed

    Kang, Joo Hyun; Chung, June-Key

    2008-06-01

    Molecular imaging includes proteomic, metabolic, cellular biologic process, and genetic imaging. In a narrow sense, molecular imaging means genetic imaging and can be called molecular-genetic imaging. Imaging reporter genes play a leading role in molecular-genetic imaging. There are 3 major methods of molecular-genetic imaging, based on optical, MRI, and nuclear medicine modalities. For each of these modalities, various reporter genes and probes have been developed, and these have resulted in successful transitions from bench to bedside applications. Each of these imaging modalities has its unique advantages and disadvantages. Fluorescent and bioluminescent optical imaging modalities are simple, less expensive, more convenient, and more user friendly than other imaging modalities. Another advantage, especially of bioluminescence imaging, is its ability to detect low levels of gene expression. MRI has the advantage of high spatial resolution, whereas nuclear medicine methods are highly sensitive and allow data from small-animal imaging studies to be translated to clinical practice. Moreover, multimodality imaging reporter genes will allow us to choose the imaging technologies that are most appropriate for the biologic problem at hand and facilitate the clinical application of reporter gene technologies. Reporter genes can be used to visualize the levels of expression of particular exogenous and endogenous genes and several intracellular biologic phenomena, including specific signal transduction pathways, nuclear receptor activities, and protein-protein interactions. This technique provides a straightforward means of monitoring tumor mass and can visualize the in vivo distributions of target cells, such as immune cells and stem cells. Molecular imaging has gradually evolved into an important tool for drug discovery and development, and transgenic mice with an imaging reporter gene can be useful during drug and stem cell therapy development. Moreover, instrumentation

  17. An Assessment and Annotated Bibliography of Marine Bioluminescence Research: 1979-1987.

    DTIC Science & Technology

    1993-01-01

    Frontiers 32(l):4-10. 339. Kils, U. (1979). Performance of Antarctic Krill Bioluminescence in larvaceans is described. 0 Euphausia superba , at...CT11830. 128. Dera, Jerzy and Theresa Weglenska (1983). Bioluminescence of Zooplankton in the Antarctic 130. Donaldson, Thomas Q., Stevens P. Tucker...from the Pressure Changes. Naval Research Laboratory, surface to 70 m (bottom) in Ezcurra Inlet during Washington, D.C., Report 8772. the Antarctic

  18. A generalized hybrid algorithm for bioluminescence tomography

    PubMed Central

    Shi, Shengkun; Mao, Heng

    2013-01-01

    Bioluminescence tomography (BLT) is a promising optical molecular imaging technique on the frontier of biomedical optics. In this paper, a generalized hybrid algorithm has been proposed based on the graph cuts algorithm and gradient-based algorithms. The graph cuts algorithm is adopted to estimate a reliable source support without prior knowledge, and different gradient-based algorithms are sequentially used to acquire an accurate and fine source distribution according to the reconstruction status. Furthermore, multilevel meshes for the internal sources are used to speed up the computation and improve the accuracy of reconstruction. Numerical simulations have been performed to validate this proposed algorithm and demonstrate its high performance in the multi-source situation even if the detection noises, optical property errors and phantom structure errors are involved in the forward imaging. PMID:23667787

  19. A synthetic luciferin improves bioluminescence imaging in live mice

    PubMed Central

    Evans, Melanie S.; Chaurette, Joanna P.; Adams, Spencer T.; Reddy, Gadarla R.; Paley, Miranda A.; Aronin, Neil; Prescher, Jennifer A.; Miller, Stephen C.

    2014-01-01

    Firefly luciferase is the most widely used optical reporter for noninvasive bioluminescence imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI from existing luciferase reporters and enables imaging in the brain that could not be achieved with D-luciferin. PMID:24509630

  20. A synthetic luciferin improves bioluminescence imaging in live mice.

    PubMed

    Evans, Melanie S; Chaurette, Joanna P; Adams, Spencer T; Reddy, Gadarla R; Paley, Miranda A; Aronin, Neil; Prescher, Jennifer A; Miller, Stephen C

    2014-04-01

    Firefly luciferase is the most widely used optical reporter for noninvasive bioluminescence imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI with existing luciferase reporters and enables imaging in the brain that could not be achieved with D-luciferin.

  1. European ring exercise on water toxicity using different bioluminescence inhibition tests based on Vibrio fischeri, in support to the implementation of the water framework directive.

    PubMed

    Farré, Marinella; Martínez, Elena; Hernando, M-D; Fernández-Alba, Amadeo; Fritz, Johann; Unruh, Eckehardt; Mihail, Otilia; Sakkas, Vasilis; Morbey, Ana; Albanis, Triantafyllos; Brito, Fatima; Hansen, Peter D; Barceló, Damià

    2006-04-15

    An inter-laboratory comparison exercise was conducted under the European Union funded project entitled: Screening Methods for Water Data Information in Support of the Implementation of the Water Framework Directive (SWIFT-WFD) and coordinated by the Consejo Superior de Investigaciones Científicas (CSIC), in order to evaluate the reproducibility of different toxicity tests based on the bioluminescence inhibition of Vibrio fischeri, for the rapid water toxicity assessment. For the first time, this type of exercise has been organized in Europe, and using different tests based on the same principle. In this exercise, 10 laboratories from 8 countries (Austria, Cyprus, Germany, Greece, Italy, Portugal, Romania, and Spain) took place, and a total number of 360 samples were distributed. During the exercise, six series of six samples were analyzed along 5 months. Every batch of samples was composed by three real samples and three standard solutions. The real samples were: a raw influent and the effluent of a wastewater treatment plant (WWTP), and a sample from a first settlement of the WWTP spiked with a mixture of toxicant standards. A final number of 330 (91.7%) samples was analyzed, 3300 values in duplicate were collected, and the results for each sample were expressed as the 50% effective concentration (EC(50)) values calculated through five points of dilution inhibition curves, after 5 and 15min of incubation times. A statistical study was initiated using 660 results. The mean values, standard deviations (sigma), variances (sigma(2)), and upper and lower warning limits (UWL and LWL) were obtained, using the EC(50) values calculated with the result from the participating laboratories. The main objectives of this toxicity ring study were to evaluate the repeatability (r) and reproducibility (R) when different laboratories conduct the test, the influence of complex matrix samples, the variability between different tests based on the same principle, and to determine the

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  3. N-Alkylated 6'-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase: new potential scaffolds for bioluminescent assays.

    PubMed

    Woodroofe, Carolyn C; Shultz, John W; Wood, Monika G; Osterman, Jean; Cali, James J; Daily, William J; Meisenheimer, Poncho L; Klaubert, Dieter H

    2008-09-30

    A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.

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

    PubMed Central

    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 investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. Methods We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. Results The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. Conclusions The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a

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

    PubMed

    Jung, Song-yi; Willard, Scott T

    2014-01-30

    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 investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This

  6. Hydromechanical stimulation of bioluminescent plankton.

    PubMed

    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. Copyright 2002 John Wiley & Sons, Ltd.

  7. Determination of the Genetic Diversity of Different Bioluminescent Bacteria by Pulsed-Field Gel Electrophoresis (PFGE)

    PubMed Central

    Ersoy Omeroglu, Esra

    2015-01-01

    . harveyi and other strains, respectively. Conclusions: The obtained results revealed that there has been a high rate of genetic diversity in bioluminescent strains isolated from Gulf of Izmir and V. lentus and V. crassostreae strains could be also bioluminescent for the first report. At the same time, PFGE analysis of bioluminescent bacteria including four different genera and ten different species were shown for the first time by this study. It is considered that data acquired by this study will contribute evolution and mechanism of bioluminescence to further works to be done. PMID:26421141

  8. A Real-Time Non-invasive Auto-bioluminescent Urinary Bladder Cancer Xenograft Model.

    PubMed

    John, Bincy Anu; Xu, Tingting; Ripp, Steven; Wang, Hwa-Chain Robert

    2017-02-01

    The study was to develop an auto-bioluminescent urinary bladder cancer (UBC) xenograft animal model for pre-clinical research. The study used a humanized, bacteria-originated lux reporter system consisting of six (luxCDABEfrp) genes to express components required for producing bioluminescent signals in human UBC J82, J82-Ras, and SW780 cells without exogenous substrates. Immune-deficient nude mice were inoculated with Lux-expressing UBC cells to develop auto-bioluminescent xenograft tumors that were monitored by imaging and physical examination. Lux-expressing auto-bioluminescent J82-Lux, J82-Ras-Lux, and SW780-Lux cell lines were established. Xenograft tumors derived from tumorigenic Lux-expressing auto-bioluminescent J82-Ras-Lux cells allowed a serial, non-invasive, real-time monitoring by imaging of tumor development prior to the presence of palpable tumors in animals. Using Lux-expressing auto-bioluminescent tumorigenic cells enabled us to monitor the entire course of xenograft tumor development through tumor cell implantation, adaptation, and growth to visible/palpable tumors in animals.

  9. Inhibition of bioluminescence in the living gills of the luminous fungus Mycena chlorophos by trans-4-aminocinnamic acid.

    PubMed

    Teranishi, Katsunori

    2017-06-24

    The living gills of the fungus Mycena chlorophos spontaneously emit green light. It was previously reported that trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnnamic acid are essential for the bright light production in the living gills. However, the chemical mechanisms underlying their bioluminescence are unknown. In the present study, trans-4-aminocinnamic acid was found to inhibit light production in the living gills. The concentrations of trans-4-aminocinnamic acid that inhibited the bioluminescence intensity by 50% of initial values for immature and mature gills were 0.07 μM and 4 μM, respectively. Approximately 20% of the bioluminescence intensity of the immature and mature gills was not inhibited by a further increase in the concentration of trans-4-aminocinnamic acid. Moreover, the bioluminescence that was activated by trans-4-hydroxycinnamic acid or trans-3,4-dihydroxycinnamic acid (0.01 mM) was completely inhibited by trans-4-aminocinnamic acid. Therefore, trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid functioned for the bioluminescence that was inhibited by trans-4-aminocinnamic acid. trans-4-Aminocinnamic acid strongly bound to the bioluminescence system(s) and withstood rinsing of the gills with 10 mM phosphate buffer (pH = 7), and high concentrations of trans-4-hydroxycinnamic acid (1 mM) and trans-3,4-dihydroxycinnamic acid (0.1 mM) functioned to displace trans-4-aminocinnamic acid from the bioluminescence system(s) and reactivate bioluminescence. Benzenamine, trans-cinnamic acid, trans-2-aminocinnamic acid, and trans-3-aminocinnamic acid did not inhibit bioluminescence. Therefore, the structure-specific inhibition by trans-4-aminocinnamic acid suggested that the 4-hydroxy group in trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid molecules plays a functional role in the bioluminescence reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Effect of optical tissue clearing on spatial resolution and sensitivity of bioluminescence imaging.

    PubMed

    Jansen, E Duco; Pickett, Patrick M; Mackanos, Mark A; Virostko, John

    2006-01-01

    In vivo bioluminescence imaging (BLI) is a powerful method of in vivo molecular imaging based on the use of optically active luciferase reporter genes. Although this method provides superior sensitivity relative to other in vivo imaging methods, spatial resolution is poor due to light scattering. The objective of this study was to use hyperosmotic agents to reduce the scattering coefficient and hence improve spatial resolution of the BLI method. A diffusing fiber tip was used to simulate an isotropic point source of bioluminescence emission (550 to 650 nm). Mouse skin was treated in vitro and in vivo with glycerol (50%, 30 min) and measurements of optical properties, and imaging photon counts were made before, during, and after application of glycerol to the skin sample. Glycerol application to mouse skin had little effect on the absorption coefficient but reduced the reduced scattering coefficient by more than one order of magnitude. This effect was reversible. Consequently, the spot size (i.e., spatial resolution) of the bioluminescence point source imaged through the skin decreased by a factor of 2 (550-nm light) to 3 (650-nm light) after 30 min. Simultaneously, an almost twofold decrease in the amount of light detected by the BLI system was observed, despite the fact that total transmission increased 1.7 times. We have shown here that multiply scattered light is responsible for both observations. We have shown that applying a hyperosmotic clearing agent to the skin of small rodents has the potential to improve spatial resolution of BLI owing to a reduction in the reduced scattering coefficient in the skin by one order of magnitude. However, reducing the scattering coefficient reduces the amount of light reaching the camera due to a reduction in the amount of multiply scattered light that reaches the camera aperture and thus reducing the sensitivity of the method.

  11. Bioluminescent Bacterial Imaging In Vivo

    PubMed Central

    Akin, Ali R.; O'Brien, Anne; Gao, Xuefeng; Tabirca, Sabin; Francis, Kevin P.; Tangney, Mark

    2012-01-01

    This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system. PMID:23149597

  12. Bioluminescent bacterial imaging in vivo.

    PubMed

    Baban, Chwanrow K; Cronin, Michelle; Akin, Ali R; O'Brien, Anne; Gao, Xuefeng; Tabirca, Sabin; Francis, Kevin P; Tangney, Mark

    2012-11-04

    This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

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

  15. Monitoring Neural Activity with Bioluminescence during Natural Behavior

    PubMed Central

    Naumann, Eva A.; Kampff, Adam R.; Prober, David A.; Schier, Alexander F.; Engert, Florian

    2010-01-01

    Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. Here we describe the use of bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish expressing the Ca2+-sensitive photoprotein GFP-apoAequorin (GA) in most neurons generated large and fast bioluminescent signals related to neural activity, neuroluminescence, that could be recorded continuously for many days. To test the limits of this technique, GA was specifically targeted to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Thus, our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior. PMID:20305645

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

    PubMed

    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.

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

    PubMed Central

    Marek, Paul E.; Moore, Wendy

    2015-01-01

    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. PMID:25941389

  18. Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation.

    PubMed

    Yagur-Kroll, Sharon; Bilic, Benny; Belkin, Shimshon

    2010-05-01

    Bioluminescent bacterial sensors are based upon the fusion of bacterial bioluminescence (lux) genes, acting as a reporter element, to selected bacterial stress-response gene promoters. Depending upon the nature of the promoter, the resulting constructs react to diverse types of environmental stress, including the presence of toxic chemicals, by dose-dependant light emission. Two bacterial sensors, harbouring sulA::luxCDABE and grpE::luxCDABE fusions, activated by the model chemicals nalidixic acid (NA) and ethanol, respectively, were subjected to molecular manipulations of the promoter region, in order to enhance the intensity and speed of their response and lower their detection thresholds. By manipulating the length of the promoter-containing segment (both promoters), by introducing random or specific mutations in the promoter sequence or by duplicating the promoter sequence (sulA only), major improvements in sensor performance were obtained. Improvements included significantly enhanced sensitivity, earlier response times and an increase in signal intensity. The general approaches described herein may be of general applicability for optimizing bacterial sensor performance, regardless of the sensing or reporting elements employed.

  19. Detection of a bioluminescent milky sea from space

    PubMed Central

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

    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 microalgal bloom in the surface waters. Because of their ephemeral nature and the paucity of scientific observations, an explanation of milky seas has remained elusive. Here, we report the first satellite observations of the phenomenon. An ≈15,400-km2 area of the northwestern Indian Ocean, roughly the size of the state of Connecticut, was observed to glow over 3 consecutive nights, corroborated on the first night by a ship-based account. This unanticipated application of satellite remote-sensing technology provides insights pertaining to the formation and scale of these poorly understood events. PMID:16186481

  20. Detection of a bioluminescent milky sea from space.

    PubMed

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

    2005-10-04

    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 microalgal bloom in the surface waters. Because of their ephemeral nature and the paucity of scientific observations, an explanation of milky seas has remained elusive. Here, we report the first satellite observations of the phenomenon. An approximately 15,400-km(2) area of the northwestern Indian Ocean, roughly the size of the state of Connecticut, was observed to glow over 3 consecutive nights, corroborated on the first night by a ship-based account. This unanticipated application of satellite remote-sensing technology provides insights pertaining to the formation and scale of these poorly understood events.

  1. Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays

    PubMed Central

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

    2015-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices. PMID:25084996

  2. Flexible peritoneal windows for quantitative fluorescence and bioluminescence preclinical imaging.

    PubMed

    Souris, Jeffrey S; Hickson, Jonathan A; Msezane, Lambda; Rinker-Schaeffer, Carrie W; Chen, Chin-Tu

    2013-01-01

    At present, there is considerable interest in the use of in vivo fluorescence and bioluminescence imaging to track the onset and progression of pathologic processes in preclinical models of human disease. Optical quantitation of such phenomena, however, is often problematic, frequently complicated by the overlying tissue's scattering and absorption of light, as well as the presence of endogenous cutaneous and subcutaneous fluorophores. To partially circumvent this information loss, we report here the development of flexible, surgically implanted, transparent windows that enhance quantitative in vivo fluorescence and bioluminescence imaging of optical reporters. These windows are metal and glass free and thus compatible with computed tomography, magnetic resonance imaging, positron emission tomography, and single-photon emission computed tomography; they also permit visualization of much larger areas with fewer impediments to animal locomotion and grooming than those previously described. To evaluate their utility in preclinical imaging, we surgically implanted these windows in the abdominal walls of female athymic nude mice and subsequently inoculated each animal with 1 × 10(4) to 1 × 10(6) bioluminescent human ovarian cancer cells (SKOV3ip.1-luc). Longitudinal imaging studies of fenestrated animals revealed up to 48-fold gains in imaging sensitivity relative to nonfenestrated animals, with relatively few complications, allowing wide-field in vivo visualization of nascent metastatic ovarian cancer colonization.

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

    PubMed

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

    2013-06-24

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

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

    PubMed

    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.

  5. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes

    PubMed Central

    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

  6. Analytical Applications of Bioluminescence and Chemiluminescence

    NASA Technical Reports Server (NTRS)

    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.

  7. Structural basis for the spectral difference in luciferase bioluminescence.

    PubMed

    Nakatsu, Toru; Ichiyama, Susumu; Hiratake, Jun; Saldanha, Adrian; Kobashi, Nobuyuki; Sakata, Kanzo; Kato, Hiroaki

    2006-03-16

    Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5'-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

  8. Ecology of colors of firefly bioluminescence

    SciTech Connect

    Lall, A.B.; Seliger, H.H.; Biggley, W.H.; Lloyd, J.E.

    1980-10-31

    Dark-active North American fireflies emit green bioluminescence and dusk-active species emit yellow, in general. Yellow light and yellow visual spectral sensitivity may be adaptations to increase the signal-to-noise (that is, foliage-reflected ambient light) ratio for sexual signaling during twilight. The peaks of the electroretinogram visual spectral sensitivities of four species tested, two dark- and two dusk-active, correspond with the peak of their bioluminescent emissions.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed Central

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

    2015-01-01

    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. PMID:26516295

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    Lu, Yujie; Douraghy, Ali; Machado, Hidevaldo B.; Stout, David; Tian, Jie; Herschman, Harvey; Chatziioannou, Arion F.

    2009-11-01

    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.

  14. Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents.

    PubMed

    Rakotonirainy, Malalanirina Sylvia; Arnold, Sylvia

    2008-01-01

    Fungal contamination is a major cause of deterioration in libraries and archives. Curators and conservators increasingly need rapid microbiological analyses. This paper presents a rapid detection method for the fungal contaminants on documents. A previous study showed that the calculation of energy charge, using bioluminescence ATP assays, provides a useful indicator to determinate the viability of fungal strains. We argue that this sensitive and time-saving method is better than traditional culture techniques. However, the procedure needs to be modified to make it usable for lay persons. An improved and simplified protocol is proposed here for the extraction of adenylate nucleotides (AN) from fungal spores and for their measurements. Our new procedure can detect the existence of viable fungal strains on documents, presenting suspect spots within minutes. The extraction is performed by filtration with DMSO-TE solution as extractant. The different step of the measurement of AN content is carried out successively in a single test tube instead of the three tubes necessary in the initial method. The new procedure was tested on 12 strains among those most frequently found in archives and libraries and validated on swab samples from real documents.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Bioluminescence in Dinoflagellates: Evidence that the Adaptive Value of Bioluminescence in Dinoflagellates is Concentration Dependent.

    PubMed

    Hanley, Karen A; Widder, Edith A

    2017-03-01

    Three major hypotheses have been proposed to explain why dinoflagellate bioluminescence deters copepod grazing: startle response, aposematic warning, and burglar alarm. These hypotheses propose dinoflagellate bioluminescence (A) startles predatory copepods, (B) warns potential predators of toxicity, and (C) draws the attention of higher order visual predators to the copepod's location. While the burglar alarm is the most commonly accepted hypothesis, it requires a high concentration of bioluminescent dinoflagellates to be effective, meaning the bioluminescence selective advantage at lower, more commonly observed, dinoflagellate concentrations may result from another function (e.g. startle response or aposematic warning). Therefore, a series of experiments was conducted to evaluate copepod grazing (Acartia tonsa) on bioluminescent dinoflagellates (during bioluminescent and nonbioluminescent phases, corresponding to night and day, respectively) at different concentrations (10, 1000, and 3000 cells mL(-1) ), on toxic (Pyrodinium bahamense var. bahamense) and nontoxic (Lingulodinium polyedrum) bioluminescent dinoflagellates, and in the presence of nonluminescent diatoms (Thalassiosira eccentrica). Changes in copepod ingestion rates, clearance rates, and feeding preferences as a result of these experimental factors, particularly during the mixed trails with nonluminescent diatoms, indicate there is a concentration threshold at which the burglar alarm becomes effective and below which dinoflagellate bioluminescence functions as an aposematic warning.

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

    PubMed

    Kuncová, Gabriela; Ishizaki, Takayuki; Solovyev, Andrey; Trögl, Josef; Ripp, Steven

    2016-06-15

    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.

  18. A bioluminescent arsenite biosensor designed for inline water analyzer.

    PubMed

    Prévéral, Sandra; Brutesco, Catherine; Descamps, Elodie C T; Escoffier, Camille; Pignol, David; Ginet, Nicolas; Garcia, Daniel

    2017-01-01

    Whole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter PrpoD. In the arsenite biosensor, the arsenite-inducible promoter P ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 °C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 μM arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field.

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

    PubMed

    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.

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

    SciTech Connect

    Feng Jinchao; Qin Chenghu; Jia Kebin; Han Dong; Liu Kai; Zhu Shouping; Yang Xin; Tian Jie

    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

  1. A posteriori correction for source decay in 3D bioluminescent source localization using multiview measured data

    NASA Astrophysics Data System (ADS)

    Sun, Li; Wang, Pu; Tian, Jie; Liu, Dan; Wang, Ruifang

    2009-02-01

    As a novel optical molecular imaging technique, bioluminescence tomography (BLT) can be used to monitor the biological activities non-invasively at the cellular and molecular levels. In most of known BLT studies, however, the time variation of the bioluminescent source is neglected. It gives rise to the inconsistent views during the multiview continuous wave measurement. In other words, the real measured data from different measured views come from 'different' bioluminescent sources. It could bring large errors in bioluminescence reconstruction. In this paper, a posteriori correction strategy for adaptive FEM-based reconstruction is proposed and developed. The method helps to improve the source localization considering the bioluminescent energy variance during the multiview measurement. In the method, the correction for boundary signals by means of a posteriori correction strategy, which adopts the energy ratio of measured data in the overlapping domains between the adjacent measurements as the correcting factor, can eliminate the effect of the inconsistent views. Then the adaptive mesh refinement with a posteriori error estimation helps to improve the precision and efficiency of BLT reconstruction. In addition, a priori permissible source region selection based on the surface measured data further reduces the ill-posedness of BLT and enhances numerical stability. Finally, three-dimension numerical simulations using the heterogeneous phantom are performed. The numerically measured data is generated by Monte Carlo (MC) method which is known as the Gold standard and can avoid the inverse crime. The reconstructed result with correction shows more accuracy compared to that without correction.

  2. A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

    PubMed

    Du, Jiao; Xu, Qinfeng; Lu, Xiaoquan; Zhang, Chun-yang

    2014-08-19

    Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

  3. Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

    PubMed

    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.

  4. Copper toxicity to bioluminescent Nitrosomonas europaea in soil is explained by the free metal ion activity in pore water.

    PubMed

    Ore, S; Mertens, J; Brandt, K K; Smolders, E

    2010-12-01

    The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu(2+) ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10(-5) to 10(-7) M and increased with increasing activities of H(+), Ca(2+) and Mg(2+) according to the BLM concept. The solution based Cu(2+) EC50 values of N. europaea in six contaminated soils ranged 2 × 10(-6) to 2 × 10(-9) M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu(2+) EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu(2+) in solution represents the toxic species to this bacterium.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    Sun, Yanwen; Connor, Michael G; Pennington, Jarrod M; Lawrenz, Matthew B

    2012-01-01

    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.

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

    PubMed Central

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

    2014-01-01

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

  8. Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging

    PubMed Central

    Inagaki, Shigenori; Tsutsui, Hidekazu; Suzuki, Kazushi; Agetsuma, Masakazu; Arai, Yoshiyuki; Jinno, Yuka; Bai, Guirong; Daniels, Matthew J.; Okamura, Yasushi; Matsuda, Tomoki; Nagai, Takeharu

    2017-01-01

    We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible. PMID:28205521

  9. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    PubMed Central

    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 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. PMID:18256736

  10. In vivo cell tracking with bioluminescence imaging.

    PubMed

    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.

  11. Optimisation of acquisition time in bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  12. Bioluminescent imaging of bacteria during mouse infection.

    PubMed

    Warawa, Jonathan M; Lawrenz, Matthew B

    2014-01-01

    Diagnostic imaging is a powerful tool that has recently been applied towards the study of infectious diseases. Optical imaging of bioluminescently labeled bacteria in infected animals allows for real-time analysis of bacterial proliferation and dissemination during infection without sacrificing the animal. Imaging also allows for tracking of disease progression in an individual subject over time, has the potential to reveal previously overlooked sites of infection, and reduces the number of research animals used in pathogenesis studies. Here, we describe the use of a deep-cooled CCD camera imager to record light emitted from bacteria during infection. We also describe the process of correlating bioluminescence to bacterial numbers by ex vivo imaging of necropsied tissues. Together these techniques can be used to estimate bacterial burdens in host tissues both in vivo and ex vivo using bioluminescent imaging.

  13. A Multichannel Bioluminescence Determination Platform for Bioassays.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    Chu, Jun; Oh, Young-Hee; 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.

    2016-01-01

    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 due 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

  16. Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

    PubMed

    Trezzani, I; Nadri, M; Dorel, C; Lejeune, P; Bellalou, J; Lieto, J; Hammouri, H; Longin, R; Dhurjati, P

    2003-01-01

    On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

  17. Use of bioluminescence markers to detect Pseudomonas spp. in the Rhizosphere

    SciTech Connect

    De Weger, L.A.; Lugtenberg, B.J.J. ); Dunbar, P.; Sayler, G.S. ); Mahafee, W.F. )

    1991-12-01

    The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10{sup 3} to 10{sup 4} CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than {beta}-galactosidase-based systems.

  18. Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

    PubMed Central

    de Weger, Letty A.; Dunbar, Paul; Mahafee, Walter F.; Lugtenberg, Ben J. J.; Sayler, Gary S.

    1991-01-01

    The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 103 to 104 CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems. Images PMID:16348610

  19. The power of bioluminescence imaging in understanding host-pathogen interactions.

    PubMed

    Suff, Natalie; Waddington, Simon N

    2017-08-15

    Infectious diseases are one of the leading causes of death worldwide. Modelling and understanding human infection is imperative to developing treatments to reduce the global burden of infectious disease. Bioluminescence imaging is a highly sensitive, non-invasive technique based on the detection of light, produced by luciferase-catalysed reactions. In the study of infectious disease, bioluminescence imaging is a well-established technique; it can be used to detect, localize and quantify specific immune cells, pathogens or immunological processes. This enables longitudinal studies in which the spectrum of the disease process and its response to therapies can be monitored. Light producing transgenic rodents are emerging as key tools in the study of host response to infection. Here, we review the strategies for identifying biological processes in vivo, including the technology of bioluminescence imaging and illustrate how this technique is shedding light on the host-pathogen relationship. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Monitoring of bacterial contamination of dental unit water lines using adenosine triphosphate bioluminescence.

    PubMed

    Watanabe, A; Tamaki, N; Yokota, K; Matsuyama, M; Kokeguchi, S

    2016-12-01

    Bacterial contamination of dental unit waterlines (DUWLs) was evaluated using ATP bioluminescence analysis and a conventional culture method. Water samples (N=44) from DUWLs were investigated for heterotrophic bacteria by culture on R2A agar, which gave counts ranging from 1.4×10(3) to 2.7×10(5) cfu/mL. The ATP bioluminescence results for DUWL samples ranged from 6 to 1189 relative light units and could be obtained within 1min; these correlated well with the culture results (r=0.727-0.855). We conclude that the results of the ATP bioluminescence assay accurately reflect the results of conventional culture-based testing. This method is potentially useful for rapid and simple monitoring of DUWL bacterial contamination. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  1. Antioxidant assay using genetically engineered bioluminescent Escherichia coli

    NASA Astrophysics Data System (ADS)

    Bartolome, Amelita; Macalino, Bernadette; Pastoral, Ian Lemuel; Sevilla, Fortunato, III

    2006-02-01

    A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (E. coli DPD2511) is described. The strain harbors the plasmid pKatG::luxCDABE and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of E. coli DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.

  2. Bioluminescence Tomography: Biomedical Background, Mathematical Theory, and Numerical Approximation 1)

    PubMed Central

    Han, Weimin; Wang, Ge

    2010-01-01

    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. PMID:20617105

  3. Bioluminescent indicator applicable to membrane voltage recording in various excitable cell types

    NASA Astrophysics Data System (ADS)

    Inagaki, Shigenori; Nagai, Takeharu

    2017-04-01

    Here, we report a world-first bioluminescent indicator for membrane voltage, LOTUS-V. Since it is able to reveal voltage dynamics without external light source, LOTUS-V serves high contrast voltage imaging free from the effect of autofluorescence, suggesting its great versatility in the wide range of bioscience.

  4. Real time detection of live microbes using a highly sensitive bioluminescent nitroreductase probe.

    PubMed

    Wong, Roger H F; Kwong, Thomas; Yau, Kwok-Hei; Au-Yeung, Ho Yu

    2015-03-14

    A highly sensitive and selective nitroreductase probe, showing a rapid and strong bioluminescence enhancement (>100-fold in 5 minutes), and its initial application in the real time detection of both Gram positive and Gram negative live bacteria and monitoring of their growth has been reported.

  5. Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization

    PubMed Central

    Rodea, Gerardo E.; Montiel-Infante, Francisco X.; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A.; Espinosa-Mazariego, Karina; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2017-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging. PMID:28560186

  6. Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization.

    PubMed

    Rodea, Gerardo E; Montiel-Infante, Francisco X; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A; Espinosa-Mazariego, Karina; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2017-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging.

  7. Bioluminescence tomography guided radiation therapy for preclinical research

    PubMed Central

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

    2015-01-01

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

  8. Noninvasive visualization of retinoblastoma growth and metastasis via bioluminescence imaging.

    PubMed

    Ji, Xunda; Cheng, Lin; Wei, Fang; Li, Huiming; Wang, Mengyun; Tian, Yuhua; Chen, Xiafang; Wang, Yufei; Wolf, Frank; Li, Chuanyuan; Huang, Qian

    2009-12-01

    To establish human retinoblastoma (RB) animal models that allow sensitive, noninvasive and continuous monitoring of tumor growth and metastasis in vivo. The human RB tumor cell lines HXO-Rb44 and Y79 were engineered to express a fusion reporter gene allowing for bioluminescence and fluorescence imaging. Intraocular and metastatic tumors were induced in immunodeficient nude mice by injection of bioluminescent RB cells into eye compartments and into the left ventricle or tail vein. The growth kinetics of intraocular and metastatic tumors was quantitatively and continuously monitored via bioluminescence imaging (BLI). Intraocular injection of HXO-Rb44-GFP-luc cells resulted in 100%, 80%, and 80% successful RB tumor development in the anterior chamber, vitreal cavity and subretinal space, respectively. The subretinal injection of Y79-GFP-luc cells resulted in 100% tumor development. BLI signal intensity correlated with the number of tumor cells injected as well as the weight of the tumor-bearing eyes. After bilateral subretinal injection of HXO-Rb44-GFP-luc cells, one of six RB tumor mice developed brain metastasis. Intracardiac injection of HXO-Rb44-GFP-luc cells resulted in metastatic disease in 9 of 15 nude mice, whereas tail vein injection resulted in metastasis in 1 of 16. Metastases were developed in multiple organs, including lymph nodes, bone, and brain, resembling the metastatic profile in patients with RB. BLI allowed sensitive, noninvasive, and quantitative localization and monitoring of intraocular and metastatic RB tumor growth in vivo and thus may be a useful tool to study RB biology as well as anti-RB therapies.

  9. Bioluminescent bioreporter integrated circuit detection methods

    DOEpatents

    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. Bioluminescence imaging of bone metastasis in rodents.

    PubMed

    Snoeks, Thomas J A; van Beek, Ermond; Que, Ivo; Kaijzel, Eric L; Löwik, Clemens W G M

    2012-01-01

    Optical imaging is a valuable technique for visualizing and quantifying biological processes in living -organisms. Optical imaging can be divided into two main imaging modalities: bioluminescence imaging and fluorescence imaging. This chapter describes the use of these imaging techniques to image tumour cells in mouse models of cancer and to detect early bone metastasis.

  11. High-Resolution Measurements of Coastal Bioluminescence

    DTIC Science & Technology

    2006-09-30

    seen at the canyon edge. The bioluminescence signal confirms that this is biological, and likely a swarm of krill , which it also detects high levels...lifesci.ucsb.edu/~biolum/ Invited talks, Outreach articles: Sep. 2006. Science Year 2007. Photos and research discussion in Worldbook supplement

  12. Bioluminescence for determining energy state of plants

    NASA Technical Reports Server (NTRS)

    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.

  13. Bioluminescence: from chemical bonds to photons.

    PubMed

    Hastings, J W

    1975-01-01

    The biological transformation of chemical to photic energy involves an enzyme-mediated chemiluminescent reaction, in which one of the products exists in an electronically excited state, emitting a photon as it returns to the ground state. The colour of bioluminescence differs in different organisms, ranging from the deep blue (460 nm) of certain crustacea, through the bluish green (490 nm) of some bacteria, the green (530 nm) of mushrooms to the red (about 600 nm) of the railroad worm. In one case, energy transfer has been demonstrated from the enzyme system to material that emits light with a longer wavelength. The energies involved range from about 165 to 250 kJ/einstein (40 to 60 kcal/einstein). Boyle first showed that air was involved in bioluminescence in 1668 in his experiments with an air pump. Over the past 100 years, it has become clear that most if not all bioluminescent systems require molecular oxygen. The recent isolation and characterization of an oxygen-containing (peroxide) enzyme intermediate from the bacterial system is described and a reaction mechanism is postulated. This scheme is compared with other hypothetical mechanisms, in particular those involving a four-membered ring intermediate, a dioxetane, in which the simultaneous cleavage of two bonds leaves one product in an excited state. I shall discuss the special role of luciferases in bioluminescence, especially in flashing mechanisms involving 'precharged' intermediates.

  14. A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

    PubMed

    Choi, Sue Hyung; Gu, Man Bock

    2002-05-01

    A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.

  15. Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence.

    PubMed

    Sharp, Natasha J; Vandamm, Joshua P; Molineux, Ian J; Schofield, David A

    2015-05-01

    Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wβ with genes encoding bacterial luciferase (luxA and luxB) to create a "light-tagged" reporter (Wβ::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wβ::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 10(3) CFU/g after 7 h of enrichment, improving to 3.2 × 10(2) CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

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

    PubMed

    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.

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

    PubMed Central

    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

  18. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions.

    PubMed

    Marques, Simone M; Esteves da Silva, Joaquim C G

    2009-01-01

    Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH(2)), with ATP-Mg(2+) generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH(2)-AMP); the second is the oxidation and decarboxylation of LH(2)-AMP to oxyluciferin, the light emitter, producing CO(2), AMP, and photons of yellow-green light (550- 570 nm). In a dark reaction LH(2)-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH(2)-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3'-phosphate chain plus an intact terminal pyrophosphate moiety. The LH(2) stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH(2) or L-LH(2), has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

  19. Effect of chemical sanitizing agents on ATP bioluminescence measurements.

    PubMed

    Green, T A; Russell, S M; Fletcher, D L

    1998-08-01

    Experiments were conducted to determine the effects of sodium hypochlorite (SH), quaternary ammonium (QA), trisodium phosphate (TSP), lactic acid (LA), hydrogen peroxide (HP), and trichlosan (TR) on ATP bioluminescence measurements. Each sanitizer was tested at three different levels and compared to a control group containing no sanitizer. ATP from three sources was analyzed, Escherichia coli, chicken blood, and a pure ATP standard. The effect of each sanitizer was reported as a percent decrease in log10 relative light units (LRLU) for the treatment groups when compared to LRLU from the control groups. SH concentrations of 30, 50, and 70 ppm and quaternary ammonium at concentrations of 10, 20, and 30 ppm had no effect on LRLU measurements, regardless of the ATP source. LA concentrations of 0.5% and higher reduced LRLU by 75%. LRLU measurements were significantly (P < or = 0.05) reduced by approximately 60% when levels of TSP exceeded 1%. HP had no effect on LRLU measurements from any of the ATP sources at 0.1%; however, at 1% HP significantly (P < or = 0.05) decreased LRLU measurements by approximately 60% for all ATP sources. TR at 0.25% had no significant effect on LRLU measurements from any of the ATP sources. TR at 0.5 and 1% reduced LRLU measurements by 30 and 50%, respectively. These results indicate that commercial sanitizers containing LA, TSP, HP, or TR may negatively affect LRLU measurements if the sanitizer is allowed to come into direct contact with the ATP bioluminescence reagents.

  20. Conditions required for the stimulation of bioluminescence activity of the genetically engineered bacteria, P. putida mt-2 KG1206, preserved by deep-freezing.

    PubMed

    Ko, K S; Kong, In Chul

    2009-03-15

    Herein the conditions required for the stimulation of bioluminescence activity in a genetically engineered strain of Pseudomonas putida mt-2 KG1206, containing the intact TOL plasmid and a constructed plasmid with the P(m)-lux gene, are reported upon. Both sodium lactate (SL) and potassium nitrate (KNO(3)) were able to stimulate the bioluminescence activity, but a greater increase was observed with nitrogen amendment. This selected stimulant was then tested on reconstituted cells that had been preserved by deep-freezing and mixed with pure inducer solution or groundwater samples. The stimulation of bioluminescence activities for deep-frozen strain was in the range of 101-238% of the control. The effect of KNO(3) was found to be dependent on the type of inducers used and the cell conditions. In general, high bioluminescence activity was observed with groundwater samples, contaminated with high inducer compounds. However, no significant correlation was observed between the bioluminescence intensity and the total inducer concentration in the environmental samples contaminated with complex mixtures with inducers. These results should be useful when other recombinant bioluminescence strains are to be used for environmental monitoring. Overall, the results of this study demonstrate the stimulant conditions for the bioluminescence activity of genetically engineered bacteria, and suggest the potential for preliminary application of this deep-frozen engineered strain in a field-ready bioassay to conveniently detect or monitor a specific group of environmental contaminants.

  1. Bioluminescence as an ecological factor during high Arctic polar night.

    PubMed

    Cronin, Heather A; Cohen, Jonathan H; Berge, Jørgen; Johnsen, Geir; Moline, Mark A

    2016-11-02

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20-40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

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

    PubMed

    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.

  3. Bioluminescence as an ecological factor during high Arctic polar night

    NASA Astrophysics Data System (ADS)

    Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

    2016-11-01

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20–40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

  4. Bioluminescence as an ecological factor during high Arctic polar night

    PubMed Central

    Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

    2016-01-01

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20–40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night. PMID:27805028

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

    PubMed Central

    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. 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. PMID:24386879

  6. Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.

    PubMed

    Ripp, S; Jegier, P; Birmele, M; Johnson, C M; Daumer, K A; Garland, J L; Sayler, G S

    2006-03-01

    To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

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

    SciTech Connect

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

    2000-03-01

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

  8. Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

    PubMed Central

    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 facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion. PMID:24193545

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

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

    2012-03-01

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

  12. Bioluminescence imaging of myeloperoxidase activity in vivo

    PubMed Central

    Gross, Shimon; Gammon, Seth T; Moss, Britney L; Rauch, Daniel; Harding, John; Heinecke, Jay W; Ratner, Lee; Piwnica-Worms, David

    2010-01-01

    The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo−/− mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions. PMID:19305414

  13. Bioluminescent assay for human lymphocyte blast transformation.

    PubMed

    Bulanova, E G; Budagyan, V M; Romanova, N A; Brovko LYu; Ugarova, N N

    1995-05-01

    One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

  14. Bioluminescence imaging of myeloperoxidase activity in vivo.

    PubMed

    Gross, Shimon; Gammon, Seth T; Moss, Britney L; Rauch, Daniel; Harding, John; Heinecke, Jay W; Ratner, Lee; Piwnica-Worms, David

    2009-04-01

    The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo(-/-) mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions.

  15. Analysis of river water by bioluminescent biotests.

    PubMed

    Kuznetsov, A M; Rodicheva, E K; Medvedeva, S E

    1999-01-01

    The bacterial bioluminescence has high sensitivity to the action of various inhibitors of biological activity. The lyophilized luminous bacteria Photobacterium phosphoreum (Microbiosensor B17 677F) and luminous strain Escherichia coli (Microbiosensor EC) from the Culture Collection IBSO were used to create bioluminescent biotests. They have been applied in ecological monitoring to determine the overall toxicity of the Yenisei and Angara Rivers and some water sources of Altai Territory. As a rule the heaviest pollution of water in studied rivers was registered near cities and settlements. The luminous bacteria biotests are simple and convenient in work, standardized and quantitative, have rapid response to actions of different substances and high sensitivity to environmental pollutants. It takes less than 30 min to do the biotest (the other biotests take 48--96 h).

  16. SU-E-J-198: Bioluminescence Monitoring of Metastatic Breast Cancer: Quantitative Assessment of Radiation Treatment Effects and Tracking of Tumor Cells.

    PubMed

    Park, J; Schmidt, T; Graves, E; Bazalova, M; Lee, J; Contag, C; Suh, T

    2012-06-01

    To evaluate radiation treatment effects on mammary carcinoma cells, quantitative photon radiance were monitored to track light-emitting cancer cells and metastasis using in vivo bioluminescence imaging. Eight female BALB/c mice aged 8 weeks were orthotopically injected with 5×104/cc 4T1 tumor cells into the abdominal mammary gland. The firefly luciferase-based bioluminescence images were acquired every 2-3 days for 1 month. Bioluminescent intensity was analyzed in average surface radiance (photons/sec/cm(2) /sr) taken in 3-dimensional bioluminescence tomography (BLT). After 1 week, single-radiation dose of 20 Gy was delivered by orthovoltage X-rays. Variation of detected bioluminescence signals emitted from molecular cancer cells was depicted on BLT images. To delineate tumor volumes according to bioluminescence intensity on anatomical images for radiation therapy, BLT images were registered with the micro computed tomography (CT) images using surface-constrained warping. Multispectral BLT images elaborated on early detection of cancer cells, characteristics of tumor growth, and metastasis for more accurate determination of internal bioluminescent sources. The radiation-treated mice having only primary tumor volumes showed 67% decrease in bioluminescent signals, while the mice with metastatic cancer cells suggested 88% reduction, as compared to the control group. Registration of BLT with CT images guided molecular cancer cells on anatomical coordinates. The BLT imaging was a useful tool to localize cancer cells and to quantify radiation response. Application of BLT led to more accurate definition of tumor volumes including molecular probe-based microscopic cancer cells. Monitoring of bioluminescence signals enables to diagnose real-time metastatic behavior of cancer cells and determine optimal radiation treatment strategies adapted to tumor characteristics. © 2012 American Association of Physicists in Medicine.

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

    ERIC Educational Resources Information Center

    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)

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

    ERIC Educational Resources Information Center

    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)

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

    NASA Astrophysics Data System (ADS)

    Jing, Cao; Jiang-an, Wang; Ronghua, Wu

    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/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.

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

    PubMed Central

    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. PMID:27694761

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

    PubMed

    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.

  2. Bioluminescence for Detection of Trace Compounds

    DTIC Science & Technology

    1990-03-08

    Photobacterium leiognathi and their expression in Escherichia coli, Qene. 54:203-210. Eberhard, A., Burlingame, A.L., Eberhard, C., Kenyon, G.L., Nealson, K.H...1985). The stimulation of bioluminescence in Photobacterium leiognathi as a potential prescreen for antitumor agents. L Antibiotics. 39:1401-1407... Photobacterium leiognatii PL721 ligated into the plasmid pACYCI84 (Figure 2), was selected for this research because of its known response to carcinogens

  3. Polymer Drag Reduction and Bioluminescence Reduction

    DTIC Science & Technology

    2007-09-30

    jet to cavitate is also reduced by the presence of trace amounts of polymer. Imaging of bioluminescence stimulated by a turbulent jet will assess...regulated by a computer-controlled pump system located downstream of the pipe. Upstream of the pipe is a tapered nozzle to assure laminar flow at the...inlet even at high flow rates. Flow rate is measured by a mass flow meter downstream of the pump and the pressure drop within the pipe is measured

  4. Classroom Based Research Report. Final Report.

    ERIC Educational Resources Information Center

    Wiecking, Kirk M.; And Others

    Resulting from a Sacramento City College (SCC) project to improve teaching and learning by directly involving faculty in research to measure student learning outcomes, this document contains reports by seven SCC faculty members on their own classroom-based research projects. "Skills-Intervention Instruction," by Linda Briggs, describes a study…

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

    PubMed

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

    2010-05-01

    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 ES114. In both strains, FNR was required for normal fumarate- and nitrate-dependent respiration. However, contrary to the report in transgenic E. coli, FNR mediated the repression of lux. ArcA represses bioluminescence, and P(arcA)-lacZ reporters showed reduced expression in fnr mutants, suggesting a possible indirect effect of FNR on bioluminescence via arcA. Finally, the fnr mutant of ES114 was not impaired in colonization of its host squid, Euprymna scolopes. This study extends the characterization of FNR to the Vibrionaceae and underscores the importance of studying lux regulation in its native background.

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

    PubMed Central

    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 ES114. In both strains, FNR was required for normal fumarate- and nitrate-dependent respiration. However, contrary to the report in transgenic E. coli, FNR mediated repression of lux. ArcA represses bioluminescence, and ParcA-lacZ reporters showed reduced expression in fnr mutants, suggesting a possible indirect effect of FNR on bioluminescence via arcA. Finally, the fnr mutant of ES114 was not impaired in colonization of its host squid, Euprymna scolopes. This study extends characterization of FNR to the Vibrionaceae and underscores the importance of studying lux regulation in its native background. PMID:20298504

  7. Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence-A Theoretical Study.

    PubMed

    Luo, Yanling; Liu, Ya-Jun

    2016-11-02

    Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light-emitting products, and 4a-hydroxy-5-hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high-level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron-transfer mechanism.

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

    PubMed

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

    2017-02-01

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

  9. Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

    PubMed

    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. Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis.

    PubMed

    Kita-Tsukamoto, Kumiko; Wada, Minoru; Yao, Katomi; Kamiya, Akiko; Yoshizawa, Susumu; Uchiyama, Nami; Kogure, Kazuhiro

    2006-03-01

    To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.

  11. Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

    PubMed Central

    Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

    2014-01-01

    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. PMID:25329534

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

    PubMed

    Thakre, Neha A; Shanware, Arti S

    2015-09-01

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

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

    PubMed

    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. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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

    PubMed

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

    2014-01-01

    Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

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

    PubMed Central

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

    2014-01-01

    Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins. PMID:24600459

  16. Single-cell bioluminescence and GFP in biofilm research

    SciTech Connect

    Palmer, R.J. Jr, Sayler, G., White, D.C.; Phiefer, C.

    1996-12-31

    Using flow cells and a combination of microscopy techniques, we can unequivocally identify single bacterial cells that express bioluminescent and fluorescent bioreporters. We have shown that, for attached cells, bioluminescence output within a bacterial strain can vary greatly from cell to cell.

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

    ERIC Educational Resources Information Center

    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,…

  18. A REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    EPA Science Inventory

    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...

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

    ERIC Educational Resources Information Center

    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,…

  20. A REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    EPA Science Inventory

    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...

  1. Nanostructured biosensor using bioluminescence quenching technique for glucose detection.

    PubMed

    Chen, Longyan; Chen, Longyi; Dotzert, Michelle; Melling, C W James; Zhang, Jin

    2017-08-22

    Most methods for monitoring glucose level require an external energy source which may limit their application, particularly in vivo test. Bioluminescence technique offers an alternative way to provide emission light without external energy source by using bioluminescent proteins found from firefly or marine vertebrates and invertebrates. For quick and non-invasive detection of glucose, we herein developed a nanostructured biosensor by applying the bioluminescence technique. Luciferase bioluminescence protein (Rluc) is conjugated with β-cyclodextrin (β-CD). The bioluminescence intensity of Rluc can be quenched by 8 ± 3 nm gold nanoparticles (Au NPs) when Au NPs covalently bind to β-CD. In the presence of glucose, Au NPs are replaced and leave far from Rluc through a competitive reaction, which results in the restored bioluminescence intensity of Rluc. A linear relationship is observed between the restored bioluminescence intensity and the logarithmic glucose concentration in the range of 1-100 µM. In addition, the selectivity of this designed sensor has been evaluated. The performance of the senor for determination of the concentration of glucose in the blood of diabetic rats is studied for comparison with that of the concentration of glucose in aqueous. This study demonstrates the design of a bioluminescence sensor for quickly detecting the concentration of glucose sensitively.

  2. Real-time bioluminescent tracking of cellular population dynamics.

    PubMed

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

    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.

  3. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    PubMed Central

    Close, Dan; Xu, Tingling; Ripp, Steven; Sayler, Gary

    2015-01-01

    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. PMID:24166372

  4. Two-phase flow cell for chemiluminescence and bioluminescence measurements

    SciTech Connect

    Mullin, J.L.; Seitz, W.R.

    1984-01-01

    A new approach to two-phase CL (chemiluminescence) measurements is reported. A magnetically stirred reagent phase is separated from the analyte phase by a dialysis membrane so that only smaller molecules can go from one phase to the other. The system is designed so that the analyte phase flows through a spiral groove on an aluminum block that is flush against the dialysis membrane. As solution flows through the spiral grove, analyte diffuses into the reagent phase where it reacts to produce light. A simple model is developed to predict how this system will behave. Experimentally, the system is evaluated by using the luminol reaction catalyzed by peroxidase, the firefly reaction, and the bacterial bioluminescence reaction. 10 references, 4 tables, 6 figures.

  5. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    SciTech Connect

    Close, Dan; Sayler, Gary Steven; Xu, Tingting; Ripp, Steven Anthony

    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.

  6. Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

    PubMed Central

    Nakajima, Yoshihiro; Yamazaki, Tomomi; Nishii, Shigeaki; Noguchi, Takako; Hoshino, Hideto; Niwa, Kazuki; Viviani, Vadim R.; Ohmiya, Yoshihiro

    2010-01-01

    We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc. PMID:20368807

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence.

    PubMed

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

    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 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.

  9. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces

    PubMed Central

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-01-01

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate “cleanliness” using a sampling area–adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm2, 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm2) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm2, which corresponded to culture-assay levels of <2.5 CFU/cm2. An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate. PMID:27294944

  10. Analysis of the disagreement between automated bioluminescence-based and culture methods for detecting significant bacteriuria, with proposals for standardizing evaluations of bacteriuria detection methods.

    PubMed Central

    Nichols, W W; Curtis, G D; Johnston, H H

    1982-01-01

    A fully automated method for detecting significant bacteriuria is described which uses firefly luciferin and luciferase to detect bacterial ATP in urine. The automated method was calibrated and evaluated, using 308 urine specimens, against two reference culture methods. We obtained a specificity of 0.79 and sensitivity of 0.75 using a quantitative pour plate reference test and a specificity of 0.79 and a sensitivity of 0.90 using a semiquantitative standard loop reference test. The majority of specimens negative by the automated test but positive by the pour plate reference test were specimens which grew several bacterial species. We suggest that such disagreement was most likely for urine containing around 10(5) colony-forming units per ml (the culture threshold of positivity) and that these specimens were ones contaminated by urethral or vaginal flora. We propose standard procedures for calibrating and evaluating rapid or automated methods for the detection of significant bacteriuria and have analyzed our results using these procedures. We recommend that identical analyses should be reported for other evaluations of bacteriuria detection methods. PMID:6808012

  11. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces.

    PubMed

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-06-09

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate "cleanliness" using a sampling area-adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm², 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm²) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm², which corresponded to culture-assay levels of <2.5 CFU/cm². An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate.

  12. PMIS: Data Base Design Report

    ERIC Educational Resources Information Center

    Fiddleman, Richard; Gorman, Michael M.

    1972-01-01

    PMIS is a computer-based planning and management information system for local school districts. This report centers on the PMIS data bases that contain school system data by reviewing the major phases involved in their creation, explaining the factors that caused the unique orientation of the data bases, reviewing the two tasks that comprise the…

  13. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    NASA Astrophysics Data System (ADS)

    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. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  15. In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

    PubMed Central

    Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

    2016-01-01

    Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

  16. Ultrasound-modulated bioluminescence tomography

    NASA Astrophysics Data System (ADS)

    Bal, Guillaume; Schotland, John C.

    2014-03-01

    We propose a method to reconstruct the density of a luminescent source in a highly scattering medium from ultrasound-modulated optical measurements. Our approach is based on the solution to a hybrid inverse source problem for the diffusion equation.

  17. High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

    PubMed

    Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

    2007-02-01

    To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

  18. A simple bioluminescent method for measuring d-amino acid oxidase activity† †Electronic supplementary information (ESI) available: Experimental methods, UV-vis data. See DOI: 10.1039/c4cc08145e Click here for additional data file.

    PubMed Central

    Bailey, T. Spencer; Donor, Micah T.; Naughton, Sean P.

    2015-01-01

    d-Amino acid oxidase (DAO) plays important roles in regulating d-amino acid neurotransmitters and was recently identified as a key enzyme integral to hydrogen sulfide production from d-Cys. We report here the development of a simple biocompatible, bioluminescent method for measuring DAO activity based on the highly selective condensation of d-Cys with 6-hydroxy-2-cyanobenzothiazole (CBT-OH) to form d-luciferin. PMID:25408176

  19. Estimating the toxicities of organic chemicals to bioluminescent bacteria and activated sludge.

    PubMed

    Ren, Shijin; Frymier, Paul D

    2002-10-01

    Toxicity assays based on bioluminescent bacteria have several advantages including a quick response and an easily measured signal. The Shk1 assay is a procedure for wastewater toxicity testing based on the bioluminescent bacterium Shk1. Using the Shk1 assay, the toxicity of 98 organic chemicals were measured and EC50 values were obtained. Quantitative structure-activity relationship (QSAR) models based on the logarithm of the octanol-water partition coefficient (log(Kow)) were developed for individual groups of organic chemicals with different functional groups. The correlation coefficients for different groups of organic compounds varied between 0.69 and 0.99. An overall QSAR model without discriminating the functional groups, which can be used for a quick estimate of the toxicities of organic chemicals, was also developed and model predictions were compared to experimental data. The model accuracy was found to be one order of magnitude from the observed values.

  20. Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6

    PubMed Central

    Rader, Bethany A.; Stabb, Eric V.; Mandel, Mark J.

    2015-01-01

    ABSTRACT Bacterial bioluminescence is taxonomically restricted to certain proteobacteria, many of which belong to the Vibrionaceae. In the most well-studied cases, pheromone signaling plays a key role in regulation of light production. However, previous reports have indicated that certain Photobacterium strains do not use this regulatory method for controlling luminescence. In this study, we combined genome sequencing with genetic approaches to characterize the regulation of luminescence in Photobacterium leiognathi strain KNH6, an extremely bright isolate. Using transposon mutagenesis and screening for decreased luminescence, we identified insertions in genes encoding components necessary for the luciferase reaction (lux, lum, and rib operons) as well as in nine other loci. These additional loci encode gene products predicted to be involved in the tricarboxylic acid (TCA) cycle, DNA and RNA metabolism, transcriptional regulation, and the synthesis of cytochrome c, peptidoglycan, and fatty acids. The mutagenesis screen did not identify any mutants with disruptions of predicted pheromone-related loci. Using targeted gene insertional disruptions, we demonstrate that under the growth conditions tested, luminescence levels do not appear to be controlled through canonical pheromone signaling systems in this strain. IMPORTANCE Despite the long-standing interest in luminous bacteria, outside a few model organisms, little is known about the regulation and function of luminescence. Light-producing marine bacteria are widely distributed and have diverse lifestyles, suggesting that the control and significance of luminescence may be similarly diverse. In this study, we apply genetic tools to the study of regulation of light production in the extremely bright isolate Photobacterium leiognathi KNH6. Our results suggest an unusual lack of canonical pheromone-mediated control of luminescence and contribute to a better understanding of alternative strategies for regulation of a

  1. Quantitative bioluminescent detection of bacteria

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1976-01-01

    Phosphoflavins in sample are measured using photobacterial luciferase assay technique for flavin mononucleotide (FMN). Boiling perchloric acid is used to rupture cells to free bound flavin and to hydrolyze flavin adenine dinucleotide to FMN. Base-stabilized water solution of sodium borohydride is used as reactant.

  2. Marine Bioluminescence: Mechanisms and Evaluation

    DTIC Science & Technology

    2001-09-30

    response curve is being created based on the sensitivity changes of the BL system at the different concentrations of CD tested. Also being tested is the...fusiformis cells were exposed to this drug and then their ability to produce BL in response to fluid shear was compared with control cells. A dose

  3. In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

    PubMed

    Cronin, Michelle; Akin, Ali R; Francis, Kevin P; Tangney, Mark

    2016-01-01

    This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.

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

    PubMed

    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.

  5. Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.

    PubMed

    Branchini, Bruce R; Behney, Curran E; Southworth, Tara L; Fontaine, Danielle M; Gulick, Andrew M; Vinyard, David J; Brudvig, Gary W

    2015-06-24

    Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

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

    PubMed

    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.

  7. Spectroscopic studies of the light-color modulation mechanism of firefly (beetle) bioluminescence.

    PubMed

    Hirano, Takashi; Hasumi, Yosuke; Ohtsuka, Kazuhiro; Maki, Shojiro; Niwa, Haruki; Yamaji, Minoru; Hashizume, Daisuke

    2009-02-18

    To reveal the light-color modulation mechanism of firefly (beetle) bioluminescence, we investigated the spectroscopic properties of the phenolate anion 1-O(-) generated from 5,5-dimethyloxyluciferin (1-OH) using various base/solvent combinations. Phenolate anion 1-O(-) is a model compound for the keto form of wild-type oxyluciferin phenolate anion (OL(-)), which is postulated to be the emitter of the bioluminescence. The fluorescence maxima of 1-O(-) were found to depend on the base/solvent combination used, and they varied in the range 541-640 nm, which covers the almost whole range of the bioluminescence emission maximum. In a polar solvent, where (1)(1-O(-))* and the countercation (the conjugate acid of a base) make a solvent-separated ion pair or a free ion couple, the emission maxima of 1-O(-) were found to be modulated by the solvent polarity. In a less polar solvent, where (1)(1-O(-))* and the countercation are formed as a contact ion pair, the strength of the covalent character of the O8'...H bond between (1)(1-O(-))* and the countercation is operative. The effect of the base/solvent combination on the emission properties of (1)(1-O(-))* was also verified using fluorescence lifetime measurements and density functional theory calculations on 1-O(-) and its ion-pair models. On the basis of these results, we propose the following light-color modulation mechanism: (1) the light emitter is the excited singlet state of OL(-) [(1)(OL(-))*], and (2) light emission from (1)(OL(-))* is modulated by the polarity of the active-site environment of a luciferase and the degree of covalent character of the O8'...H bond between (1)(OL(-))* and a protonated basic moiety in the active site. Mechanisms for variation of the bioluminescence colors and their applications are discussed.

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

    PubMed

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

    2014-08-19

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

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

    PubMed

    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.

  10. 106 ASSESSMENT OF ANTI-BACTERIAL EFFECTS OF PEGYLATED SILVER-COATED CARBON NANOTUBES ON CAUSATIVE BACTERIA OF BOVINE INFERTILITY USING BIOLUMINESCENCE IMAGING SYSTEM.

    PubMed

    Park, S; Chaudhari, A A; Pillai, S; Singh, S R; Willard, S T; Ryan, P L; Feugang, J M

    2016-01-01

    OD values did not always reflect bacteria concentrations, and varied according to nanotube concentrations. No significant differences in anti-bacterial activities were revealed between pSWCNTs-Ag and SWCNTs-Ag based on OD values during 6h of incubation (P>0.05); meanwhile, pSWCNTs-Ag nanotubes exhibited stronger anti-bacterial effects than SWCNTs-Ag during the same period using BLI (P<0.05). In summary, we confirmed previous reports showing dose-dependent eliminations of pathogenic bacteria by silver nanotubes. Pegylated nanotubes exhibited high anti-bacterial activity compared to non-pegylated nanotubes. Bioluminescence imaging system revealed superior resolution to enable precise investigation of anti-bacterial kinetics of silver nanotubes. This feature could be useful for the study of bacterial infections that impair livestock fertility.

  11. [Use of the bioluminescent method for the determination of bacterial adenosinetriphosphate (ATP-metry) in microbiology].

    PubMed

    Kuzikov, A N; Bondarenko, V M; Latkin, A T

    2003-01-01

    The attention of a wide circle of specialists has recently been attracted by different methods for rapid determination of pathogenic microorganisms in biological specimens, environmental objects and foodstuffs, as well as in cases of possible acts of bioterrorism. In this respect the bioluminescent method for determination of adenosine triphosphate (ATP) contained in microbial cells is of interest. The method is based on the interaction ATP, luciferase and luciferin, accompanied by giving off energy in the form of light emission. When compared with routine methods, the use of this method considerably reduces the duration of the analysis, and its high sensitivity is comparable with that of the polymerase chain reaction. In this review the data on the prospects of the practical use of the bioluminescent method of ATP-metry are presented.

  12. Trace metals in arcid clam Scapharca inaequivalvis: effects of molluscan extracts on bioluminescent bacteria.

    PubMed

    Girotti, S; Bolelli, L; Fini, F; Monari, M; Andreani, G; Isani, G; Carpené, E

    2006-10-01

    The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the "bioactive" metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

  13. Bioluminescence-Activated Deep-Tissue Photodynamic Therapy of Cancer

    PubMed Central

    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/cm2 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. PMID:26000054

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

    PubMed

    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.

  15. Digital spectral separation methods and systems for bioluminescence imaging.

    PubMed

    Wang, Ge; Shen, Haiou; Liu, Ying; Cong, Alex; Cong, Wenxiang; Wang, Yue; Dubey, Purnima

    2008-02-04

    We propose a digital spectral separation (DSS) system and methods to extract spectral information optimally from a weak multi-spectral signal such as in the bioluminescent imaging (BLI) studies. This system utilizes our newly invented spatially-translated spectral-image mixer (SSM), which consists of dichroic beam splitters, a mirror, and a DSS algorithm. The DSS approach overcomes the shortcomings of the data acquisition scheme used for the current BLI systems. Primarily, using our DSS scheme, spectral information will not be filtered out. Accordingly, truly parallel multi-spectral multi-view acquisition is enabled for the first time to minimize experimental time and optimize data quality. This approach also permits recovery of the bioluminescent signal time course, which is useful to study the kinetics of multiple bioluminescent probes using multi-spectral bioluminescence tomography (MSBT).

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

    PubMed Central

    Bussey, D M; Tsuji, K

    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 encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined. PMID:3954348

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

    PubMed Central

    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. PMID:27594981

  18. Bioluminescence Assays for Monitoring Chondrogenic Differentiation and Cartilage Regeneration.

    PubMed

    Je, Hyeon Jeong; Kim, Min Gu; Kwon, Hyuck Joon

    2017-06-06

    Since articular cartilage has a limited regeneration potential, for developing biological therapies for cartilage regeneration it is important to study the mechanisms underlying chondrogenesis of stem cells. Bioluminescence assays can visualize a wide range of biological phenomena such as gene expression, signaling, metabolism, development, cellular movements, and molecular interactions by using visible light and thus contribute substantially to elucidation of their biological functions. This article gives a concise review to introduce basic principles of bioluminescence assays and applications of the technology to visualize the processes of chondrogenesis and cartilage regeneration. Applications of bioluminescence assays have been highlighted in the methods of real-time monitoring of gene expression and intracellular levels of biomolecules and noninvasive cell tracking within animal models. This review suggests that bioluminescence assays can be applied towards a visual understanding of chondrogenesis and cartilage regeneration.

  19. Bioluminescence Assays for Monitoring Chondrogenic Differentiation and Cartilage Regeneration

    PubMed Central

    Je, Hyeon Jeong; Kim, Min Gu; Kwon, Hyuck Joon

    2017-01-01

    Since articular cartilage has a limited regeneration potential, for developing biological therapies for cartilage regeneration it is important to study the mechanisms underlying chondrogenesis of stem cells. Bioluminescence assays can visualize a wide range of biological phenomena such as gene expression, signaling, metabolism, development, cellular movements, and molecular interactions by using visible light and thus contribute substantially to elucidation of their biological functions. This article gives a concise review to introduce basic principles of bioluminescence assays and applications of the technology to visualize the processes of chondrogenesis and cartilage regeneration. Applications of bioluminescence assays have been highlighted in the methods of real-time monitoring of gene expression and intracellular levels of biomolecules and noninvasive cell tracking within animal models. This review suggests that bioluminescence assays can be applied towards a visual understanding of chondrogenesis and cartilage regeneration. PMID:28587284

  20. Shedding light on bioluminescence regulation in Vibrio fischeri.

    PubMed

    Miyashiro, Tim; Ruby, Edward G

    2012-06-01

    The bioluminescence emitted by the marine bacterium Vibrio fischeri is a particularly striking result of individual microbial cells co-ordinating a group behaviour. 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 autoinduction behaviour. The Hawaiian squid Euprymna scolopes forms a natural symbiosis with V. fischeri, and utilizes the symbiont-derived bioluminescence for certain nocturnal behaviours, such as counterillumination. Recent work suggests that the tissue with which V. fischeri associates not only can detect bioluminescence but may also use this light to monitor the V. fischeri population. © 2012 Blackwell Publishing Ltd.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

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

  3. Bioluminescence Imaging of Clavibacter michiganensis subsp. michiganensis Infection of Tomato Seeds and Plants ▿

    PubMed Central

    Xu, Xiulan; Miller, Sally A.; Baysal-Gurel, Fulya; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Rajashekara, Gireesh

    2010-01-01

    Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the

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

    PubMed

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

    2015-07-01

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

  5. Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of tomato seeds and plants.

    PubMed

    Xu, Xiulan; Miller, Sally A; Baysal-Gurel, Fulya; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Rajashekara, Gireesh

    2010-06-01

    Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the

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

    PubMed

    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. © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.

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

    PubMed Central

    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

  8. Profiling the biological effects of wastewater samples via bioluminescent bacterial biosensors combined with estrogenic assays.

    PubMed

    Bazin, Ingrid; Seo, Ho Bin; Suehs, Carey M; Ramuz, Marc; De Waard, Michel; Gu, Man Bock

    2017-01-01

    Various water samples were successfully evaluated using a panel of different recombinant bioluminescent bacteria and estrogenic activity analysis. The bioluminescent bacteria strains induced by oxidative (superoxide radical or hydroxyl radical), protein damage, cell membrane damage, or cellular toxicity were used. Estrogenic activities were examined by using the yeast strain BY4741, which carries the β-galactosidase reporter gene under the control of the estrogen-responsive element (ERE). A total of 14 samples from three wastewater treatment plants, one textile factory, and seawater locations in Tunisia were analyzed. A wide range of bio-responses were described. Site/sample heterogeneity was prevalent, in combination with generally high relative bioluminescence scores for oxidative stress (OH•). Estrogenic activity was detected at all sites and was particularly elevated at certain sites. Our perspectives include the future exploration of the variation detected in relation to treatment plant operations and environmental impacts. In conclusion, this new multi-experimental method can be used for rapid bio-response profile monitoring and the evaluation of environmental samples spanning a wide range of domains. This study confirms that bio-reactive wastewater treatment plant (WWTP) effluents are discharged into seawater, where they may impact coastal populations.

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

    PubMed

    Wu, Nan; Dacres, Helen; Anderson, Alisha; Trowell, Stephen C; Zhu, Yonggang

    2014-01-01

    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. 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. 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. 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.

  10. Biological water quality monitoring using chemiluminescent and bioluminescent techniques

    NASA Technical Reports Server (NTRS)

    Thomas, R. R.

    1978-01-01

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

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

    PubMed

    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.

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

    PubMed

    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. Bioluminescence Imaging of Chlamydia muridarum Ascending Infection in Mice

    PubMed Central

    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. PMID:24983626

  14. Surface-Tunable Bioluminescence Resonance Energy Transfer via Geometry-Controlled ZnO Nanorod Coordination.

    PubMed

    Lim, Jun Hyung; Park, Geun Chul; Lee, Seung Muk; Lee, Jung Heon; Lim, Butaek; Hwang, Soo Min; Kim, Jung Ho; Park, Hansoo; Joo, Jinho; Kim, Young-Pil

    2015-07-01

    The use of ZnO nanorods (NRs) as an effective coordinator and biosensing platform to create bioluminescence resonance energy transfer (BRET) is reported. Herein, a hydrothermal approach is applied to obtain morphologically controlled ZnO NRs, which are directly bound to luciferase (Luc) and carboxy-modified quantum dot (QD) acting as a donor-acceptor pair for BRET. BRET efficiency varies significantly with the geometry of ZnO NRs, which modulates the coordination between hexahistidine-tagged Luc (Luc-His6 ) and QD, owing to the combined effect of the total surface area consisting of (001) and (100) planes and their surface polarities. Unlike typical QD-BRET reactions with metal ions (e.g., zinc ions), a geometry-controlled ZnO NR platform can facilitate the design of surface-initiated BRET sensors without being supplemented by copious metal ions: the geometry-controlled ZnO NR platform can therefore pave the way for nanostructure-based biosensors with enhanced analytical performance. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    NASA Astrophysics Data System (ADS)

    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. Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs.

    PubMed

    Kumar, Manoj; Kovalski, Letícia; Broyles, David; Hunt, Eric A; Daftarian, Pirouz; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K

    2016-04-01

    Here we describe the design and construction of an imaging construct with high bioluminescent resonance energy transfer (BRET) efficiency that is composed of multiple quantum dots (QDs; λem = 655 nm) self-assembled onto a bioluminescent protein, Renilla luciferase (Rluc). This is facilitated by the streptavidin-biotin interaction, allowing the facile formation of a hybrid-imaging construct (HIC) comprising up to six QDs (acceptor) grafted onto a light-emitting Rluc (donor) core. The resulting assembly of multiple acceptors surrounding a donor permits this construct to exhibit high resonance energy transfer efficiency (∼64.8%). The HIC was characterized using fluorescence excitation anisotropy measurements and high-resolution transmission electron microscopy. To demonstrate the application of our construct, a generation-5 (G5) polyamidoamine dendrimer (PAMAM) nanocarrier was loaded with our HIC for in vitro and in vivo imaging. We envision that this design of multiple acceptors and bioluminescent donor will lead to the development of new BRET-based systems useful in sensing, imaging, and other bioanalytical applications.

  17. Detection of protein-protein interactions using Aequorea victoria bioluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Vinokurov, Leonid M.; Gorokhovatsky, Andrey Y.; Rudenko, Natalia V.; Marchenkov, Victor V.; Skosyrev, Vitaly S.; Arzhanov, Maxim A.; Zakharov, Mikhail V.; Burkhardt, Nils; Semisotnov, Gennady V.; Alakhov, Yuli B.

    2003-07-01

    Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon taking place in some marine coelenterates. Emission of light in these organisms involves the energy transfer between chromophores of donor luciferase and acceptor fluorescent protein. Due to the strict dependence of BRET efficiency on the inter-chromophore distance, the phenomenon has been applied to study protein-protein interactions by fusing interacting partners with either donor or acceptor proteins. Here we describe a BRET-based homogeneous protein-protein interaction assay exploiting novel donor-acceptor pair formed by photoproteins of jellyfish Aequorea victoria bioluminescent system, aequorin and green fluorescent protein enhanced variant (EGFP). Two known interacting proteins, streptavidin (SAV) and biotin carboxyl carrier protein (BCCP) were fused, respectively, with aequorin and EGFP. The fusions were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-Aequorin and BCCP-EGFP was followed by BRET between aequorin (donor) and EGFP (acceptor) resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-Aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

  18. Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein

    PubMed Central

    Hattori, Mitsuru; Haga, Sanae; Takakura, Hideo; Ozaki, Michitaka; Ozawa, Takeaki

    2013-01-01

    Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues. PMID:23690604

  19. Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein.

    PubMed

    Hattori, Mitsuru; Haga, Sanae; Takakura, Hideo; Ozaki, Michitaka; Ozawa, Takeaki

    2013-06-04

    Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.

  20. High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay.

    PubMed

    Liu, Yichin; Gerber, Raphaele; Wu, John; Tsuruda, Trace; McCarter, John D

    2008-07-01

    Silent information regulator or sirtuin (SIRT) enzymes are beta-nicotinamide adenine dinucleotide (oxidized) (NAD(+))-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD(+). The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD(+) bioluminescence assay couples NAD(+) consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD(+) consumption and peptide deacetylation was shown to be 1:1 by the NAD(+) bioluminescence assay. Kinetic parameters of peptide and NAD(+) cosubstrates and IC(50) values of standard reference inhibitors determined in either assay were similar. With robust Z' values (0.7), both assays are amenable to high-throughput screening.

  1. Evaluation of the simplified spherical harmonics approximation in bioluminescence tomography through heterogeneous mouse models.

    PubMed

    Liu, Kai; Lu, Yujie; Tian, Jie; Qin, Chenghu; Yang, Xin; Zhu, Shouping; Yang, Xiang; Gao, Quansheng; Han, Dong

    2010-09-27

    In vivo bioluminescence imaging (BLI) has played a more and more important role in biomedical research of small animals. Bioluminescence tomography (BLT) further translates the BLI optical information into three-dimensional bioluminescent source distribution, which could greatly facilitate applications in related studies. Although the diffusion approximation (DA) is one of the most widely-used forward models, higher-order approximations are still needed for in vivo small animal imaging. In this work, as a relatively accurate and higher-order approximation theory, the performance of the simplified spherical harmonics approximation (SPN) in BLT is evaluated detailedly in heterogeneous small animals. In the numerical validations, the SPN based results demonstrate better imaging quality compared with diffusion approximation heterogeneously under various source locations over wide optical domain. Although the evaluation for the effects of the optical property mismatch indicates the sensitivity of SPN is similar with DA model in the source localization, it may offer improved performance with much less artifacts. In what follows, heterogeneous experimental BLT reconstructions using in vivo mouse further evaluate the capability of the higher-order method for practical biomedical applications.

  2. Fast Monitoring of Indoor Bioaerosol Concentrations with ATP Bioluminescence Assay Using an Electrostatic Rod-Type Sampler

    PubMed Central

    Park, Ji-Woon; Park, Chul Woo; Lee, Sung Hwa; Hwang, Jungho

    2015-01-01

    A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min) of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall) was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP) bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations. PMID:25950929

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

    PubMed

    Park, Ji-Woon; Park, Chul Woo; Lee, Sung Hwa; Hwang, Jungho

    2015-01-01

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

  4. A dip-stick type biosensor using bioluminescent bacteria encapsulated in color-coded alginate microbeads for detection of water toxicity.

    PubMed

    Jung, Insup; Seo, Ho Bin; Lee, Ji-eun; Kim, Byoung Chan; Gu, Man Bock

    2014-09-21

    The use of genetically engineered bioluminescent bacteria, in which bioluminescence is induced by different modes of toxic action, represents an alternative to acute toxicity tests using living aquatic organisms (plants, vertebrates, or invertebrates) in an aqueous environment. A number of these bacterial strains have been developed, but there have been no attempts to develop a hand-held type of biosensor for monitoring or identification of toxicity. We report a facile dip-stick type biosensor using genetically engineered bioluminescent bacteria as a new platform for classification and identification of toxicity in water environments. This dip-stick type biosensor is composed of eight different optically color-coded functional alginate beads that each encapsulates a different bioluminescent bacterial strain and its corresponding fluorescent microbead. These color-coded microbeads exhibit easy identification of encapsulated microbeads, since each microbead has a different color code depending on the bioluminescent bacterial strain contained and improved cell-stability compared to liquid culture. This dip-stick type biosensor can discriminate different modes of toxic actions (i.e. DNA damage, oxidative damage, cell-membrane damage, or protein damage) of sample water tested by simply dipping the stick into the water samples. It was found that each color-coded microbead emitted distinct bioluminescence, and each dip-stick type biosensor showed different bioluminescence patterns within 2 hours, depending on the toxic chemicals contained in LB medium, tap water, or river water samples. This dip-stick type biosensor can, therefore, be widely and practically used in checking toxicity of water in the environment primarily in situ, possibly indicating the status of biodiversity.

  5. AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level

    NASA Astrophysics Data System (ADS)

    Smith, David F.; Stults, Nancy L.

    1996-04-01

    AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

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

    PubMed

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

    2009-03-01

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

  7. Bioluminescence in soybean root nodules: Demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase

    PubMed Central

    Legocki, Roman P.; Legocki, Misuk; Baldwin, Thomas O.; Szalay, Aladar A.

    1986-01-01

    Two plasmid vectors pFIT001 and pPALE001, containing luxAB genes encoding bacterial luciferase [alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] from Vibrio harveyi, have been constructed. Escherichia coli carrying derivatives of pFIT001 with DNA inserts in the unique EcoRI site located in luxB form “dark” colonies that can be readily distinguished from the bioluminescent or “bright” colonies. In contrast, promoterless pPALE001 is used as a promoter-search vector based on bioluminescence. The control and regulation of gene expression can be analyzed in vivo using promoter-luxAB fusions by a variety of simple methods, including a technique called “luxdot.” As an example, we have introduced nitrogenase nifD and nifH promoter-luxAB fusions into the Bradyrhizobium japonicum chromosome and shown symbiotically regulated bioluminescence in soybean root nodules. B. japonicum transconjugants containing a single copy per genome of the nif promoter-controlled luciferase structural genes did not produce light in free-living cultures, but the same transconjugants did express bioluminescence in root nodules that was strong enough to be detected by the naked eye. Images PMID:16593783

  8. In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model

    PubMed Central

    Massey, Shane; Johnston, Katie; Mott, Tiffany M.; Judy, Barbara M.; Kvitko, Brian H.; Schweizer, Herbert P.; Estes, D. Mark; Torres, Alfredo G.

    2011-01-01

    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 5 × 103 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. PMID:21904535

  9. Detection of DNA adducts by bioluminescence

    NASA Astrophysics Data System (ADS)

    Xu, Shunqing; Tan, Xianglin; Yao, Qunfeng; He, Min; Zhou, Yikai; Chen, Jian

    2001-09-01

    Luminescent assay for detection ATP is very sensitive with limitation of 10-17 moles. ATP using styrene oxide as a model carcinogen we currently apply a luminescence technique to detect the very low levels of carcinogen-DNA adducts in vitro and in vivo. The bioluminescent assay of DNA adducts entails three consecutive steps: digestion of modified DNA to adducted dinucleoside monophosphate and normal nucleotide are hydrolyzed to nucleosides (N) by nuclease P1 and prostatic acid phosphomonesterase (PAP); incorporation of (gamma) -P of ATP into normal nucleoside(N); detection of consumption of ATP by luminescence. This assay does not require separate manipulation because of the selective property of nuclease P1. One fmol of carcinogen- DNA adducts was detected by luminescent assay. A good correlation between results of luminescent assay and 32P-postlabeling procedures has been observed. We detect 1 adduct in 108 nucleotides for 10(mu) g DNA sample. The procedures of luminescent method is very simple and low- cost. IT appears applicable to the ultra sensitive detection of low levels of DNA adducts without radioactive isotope.

  10. Effect of halogenated fluorescent compounds on bioluminescent reactions.

    PubMed

    Kirillova, Tamara N; Gerasimova, Marina A; Nemtseva, Elena V; Kudryasheva, Nadezhda S

    2011-04-01

    The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure; hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence quenching--energy transfer processes, collisional interactions, and enzyme-dye binding--were considered. Changes of bioluminescence (BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight of halogen substituents were demonstrated. The results are discussed in terms of "dark effect of heavy halogen atom" in the process of enzyme-dye binding; hydrophobic interactions were assumed to be responsible for the effect.

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

    PubMed

    Richard, Raveesh Daniel; Bowen, Thomas R

    2017-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2001-09-01

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

  13. [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].

    PubMed

    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.

  14. Iso-luminance counterillumination drove bioluminescent shark radiation

    PubMed Central

    Claes, Julien M.; Nilsson, Dan-Eric; Straube, Nicolas; Collin, Shaun P.; Mallefet, Jérôme

    2014-01-01

    Counterilluminating animals use ventral photogenic organs (photophores) to mimic the residual downwelling light and cloak their silhouette from upward-looking predators. To cope with variable conditions of pelagic light environments they typically adjust their luminescence intensity. Here, we found evidence that bioluminescent sharks instead emit a constant light output and move up and down in the water column to remain cryptic at iso-luminance depth. We observed, across 21 globally distributed shark species, a correlation between capture depth and the proportion of a ventral area occupied by photophores. This information further allowed us, using visual modelling, to provide an adaptive explanation for shark photophore pattern diversity: in species facing moderate predation risk from below, counterilluminating photophores were partially co-opted for bioluminescent signalling, leading to complex patterns. In addition to increase our understanding of pelagic ecosystems our study emphasizes the importance of bioluminescence as a speciation driver. PMID:24608897

  15. Species-specific bioluminescence facilitates speciation in the deep sea.

    PubMed

    Davis, Matthew P; Holcroft, Nancy I; Wiley, Edward O; Sparks, John S; Leo Smith, W

    2014-01-01

    The vast darkness of the deep sea is an environment with few obvious genetic isolating barriers, and little is known regarding the macroevolutionary processes that have shaped present-day biodiversity in this habitat. Bioluminescence, the production and emission of light from a living organism through a chemical reaction, is thought to occur in approximately 80 % of the eukaryotic life that inhabits the deep sea (water depth greater than 200 m). In this study, we show, for the first time, that deep-sea fishes that possess species-specific bioluminescent structures (e.g., lanternfishes, dragonfishes) are diversifying into new species at a more rapid rate than deep-sea fishes that utilize bioluminescence in ways that would not promote isolation of populations (e.g., camouflage, predation). This work adds to our understanding of how life thrives and evolution shaped present-day biodiversity in the deep sea, the largest and arguably least explored habitat on earth.

  16. The Expanding Toolbox of In Vivo Bioluminescent Imaging

    PubMed Central

    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

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

    PubMed

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

    2017-03-01

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

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

    PubMed Central

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

    2017-01-01

    Purpose We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Methods Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. Results In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. Conclusions In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis. PMID:28278321

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2015-03-02

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

  1. [An adenosine triphosphate bioluminescence assay for detecting the number of living cells].

    PubMed

    Liu, S; Peng, Z; Wang, H; Lou, J; He, B; Tang, Q; Qiu, D

    2000-06-01

    The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.

  2. NanoLuc Reporter for Dual Luciferase Imaging in Living Animals

    PubMed Central

    Stacer, Amanda C.; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E.; Rehemtulla, Alnawaz; Luker, Gary D.

    2014-01-01

    Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  5. Aequorea victoria bioluminescence moves into an exciting new era.

    PubMed

    Kendall, J M; Badminton, M N

    1998-05-01

    Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellular signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins involved in bioluminescence of the jellyfish Aequorea victoria--aequorin and green fluorescent protein. Engineered recombinant aequorin has led to a novel technological approach to monitoring calcium signals in organelles and subcellular domains. A new generation of intracellular calcium indicators has been produced in which engineered variants of green fluorescent protein are used to probe their ionic environment using intramolecular fluorescence-resonance-energy transfer.

  6. [Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli].

    PubMed

    Ptitsyn, L R; Fatova, M A; Stepanov, A I

    1990-02-01

    Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

  7. Feasibility Study for a Compact, Multi-Purpose Bioluminescence Detector

    DTIC Science & Technology

    1998-09-30

    Symposium on Bioluminescence and Chemiluminescence. Eds. JW Hastings, LJ Kricka and PE Stanley. John Wiley & Sons Ltd, Sussex, UK. pp. 159-164...Lowenstine, M.R. Bowlby , and D.P. Cook. (1993) A new large volume bioluminescence bathyphotometer with defined turbulence excitation. Deep Sea Res. 40...and PE Stanley. John Wiley & Sons Ltd, Sussex, UK. pp. 159-164. Makemson, J.C., N.R. Fulayfil, W.L. Landry, L.M. Van Ert, C.F. Wimpee, E.A. Widder

  8. Expedient synthesis of electronically modified luciferins for bioluminescence imaging

    PubMed Central

    McCutcheon, David C.; Paley, Miranda A.; Steinhardt, Rachel C.; Prescher, Jennifer A.

    2012-01-01

    Bioluminescence imaging with luciferase enzymes requires access to light-emitting, small molecule luciferins. Here, we describe a rapid method to synthesize d-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogs. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogs produced with this approach emit light with Fluc in vitro and in live cells. Collectively, our work increases the number of substrates that can be used for bioluminescence imaging and provides a general strategy for synthesizing new collections of luciferins. PMID:22519459

  9. Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs.

    PubMed

    Cevenini, Luca; Michelini, Elisa; D'Elia, Marcello; Guardigli, Massimo; Roda, Aldo

    2013-01-01

    Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ(9)-tetrahydrocannabinol as major constituent.

  10. Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

    PubMed

    Uliczka, Frank; Pisano, Fabio; Kochut, Annika; Opitz, Wiebke; Herbst, Katharina; Stolz, Tatjana; Dersch, Petra

    2011-01-01

    A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

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

    PubMed

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

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Comsa, Daria Craita

    2008-10-01

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

  14. A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

    PubMed Central

    Sha, Jian; Rosenzweig, Jason A.; Kirtley, Michelle L.; van Lier, Christina J.; Fitts, Eric C.; Kozlova, Elena V.; Erova, Tatiana E.; Tiner, Bethany L.; Chopra, Ashok K.

    2012-01-01

    The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersina pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD50. Intranasal infection of mice with 15 LD50 of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of

  15. A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague.

    PubMed

    Sha, Jian; Rosenzweig, Jason A; Kirtley, Michelle L; van Lier, Christina J; Fitts, Eric C; Kozlova, Elena V; Erova, Tatiana E; Tiner, Bethany L; Chopra, Ashok K

    2013-02-01

    The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD(50). Intranasal infection of mice with 15 LD(50) of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24-72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy

  16. Strategy for dual-analyte luciferin imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation.

    PubMed

    Van de Bittner, Genevieve C; Bertozzi, Carolyn R; Chang, Christopher J

    2013-02-06

    In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H(2)O(2)-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H(2)O(2) and caspase 8 activity. Using these probes, chemoselective imaging of either H(2)O(2) or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H(2)O(2) and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.

  17. A Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation

    PubMed Central

    Van de Bittner, Genevieve C.; Bertozzi, Carolyn R.

    2013-01-01

    In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species (ROS), as well as a peptide-based probe, Ile-Glu-Thr-Asp-D-Cys (IETDC) which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies. PMID:23347279

  18. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    PubMed

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants.

  19. Toxicity assessment of Hanford Site wastes by bacterial bioluminescence. [Photobacter phosphoreum:a3

    SciTech Connect

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

    1991-09-01

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

  20. Molecular imaging with bioluminescence and PET reveals viral oncolysis kinetics and tumor viability

    PubMed Central

    Kuruppu, Darshini; Brownell, Anna-Liisa; Shah, Khalid; Mahmood, Umar; Tanabe, Kenneth K.

    2014-01-01

    Viral oncolysis, the destruction of cancer cells by replicating virus, is an experimental cancer therapy that continues to be explored. The treatment paradigm for this therapy involves successive waves of lytic replication in cancer cells. At current, monitoring viral titer at sites of replication requires biopsy. However, repeat serial biopsies are not practically feasible for temporal monitoring of viral replication and tumor response in patients. Molecular imaging provides a non-invasive method to identify intracellular viral gene expression in real time. We imaged viral oncolysis and tumor response to oncolysis sequentially with bioluminescence and positron emission tomography (PET), revealing the kinetics of both processes in tumor xenografts. We demonstrate that virus replication cycles can be identified as successive waves of reporter expression that occur ~2 days after the initial viral tumor infection peak. These waves correspond to virions that are released following a replication cycle. The viral and cellular kinetics were imaged with Fluc and Rluc bioluminescence reporters plus two 18F-labelled PET reporters (FHBG, 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine) and (FLT, 18F- 3′-deoxy-3-′fluorothymidine), respectively. Correlative immunohistochemistry on tumor xenograft sections confirmed in vivo results. Our findings show how PET can be used to identify virus replication cycles and for real-time measurements of intratumoral replicating virus levels. This noninvasive imaging approach has potential utility for monitoring viral oncolysis therapy in patients. PMID:24876106

  1. Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

    PubMed

    Gorokhovatsky, Andrey Yu; Marchenkov, Victor V; Rudenko, Natalia V; Ivashina, Tanya V; Ksenzenko, Vladimir N; Burkhardt, Nils; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2004-07-30

    The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

  2. Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

    DTIC Science & Technology

    2009-09-30

    potential in the coastal zone environment. OBJECTIVES Blooms of bioluminescent jellyfish , especially of Mnemiopsis leidyi, are a common occurrence... jellyfish populations are done with net collections by hand at stations weekly, monthly, or seasonally. These time scales severely limit our knowledge...the collection of both biotic and abiotic data continuously. 5 IMPACT/APPLICATIONS As incidents of jellyfish blooms, especially Mnemiopsis

  3. Bioluminescence: a fungal nightlight with an internal timer.

    PubMed

    Bechara, Etelvino J H

    2015-03-30

    A recent study shows that green light emission by Neonothopanus gardneri mushrooms, endemic to coconut forests of Northern Brazil, is controlled by a circadian clock. Furthermore, insects are attracted by the light, raising the possibility that bioluminescence functions in spore dispersal and fungal dissemination. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    Nemtseva, E. V.; Kudryasheva, N. S.

    2007-01-01

    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.

  5. Bioluminescence imaging of c-fos gene expression accompanying filial imprinting in the newly hatched chick brain.

    PubMed

    Yamaguchi, Shinji; Iikubo, Eiji; Hirose, Naoki; Kitajima, Takaaki; Katagiri, Sachiko; Kawamori, Ai; Fujii-Taira, Ikuko; Matsushima, Toshiya; Homma, Koichi J

    2010-06-01

    Bioluminescence imaging is a powerful tool for examining gene expression in living animals. Previously, we reported that exogenous DNA could be successfully delivered into neurons in the newly hatched chick brain using electroporation. Here, we show the in vivo bioluminescence imaging of c-fos promoter activity and its upregulation, which is associated with filial imprinting. The upregulation of c-fos gene expression correlated with both the strength of the chicks' approach activity to the training object and the acquisition of memory. The present technique should be a powerful tool for analyzing the time changes in neural activity of certain brain areas in real-time during memory formation, using brains of living animals.

  6. Comparisons of hybrid radiosity-diffusion model and diffusion equation for bioluminescence tomography in cavity cancer detection

    NASA Astrophysics Data System (ADS)

    Chen, Xueli; Yang, Defu; Qu, Xiaochao; Hu, Hao; Liang, Jimin; Gao, Xinbo; Tian, Jie

    2012-06-01

    Bioluminescence tomography (BLT) has been successfully applied to the detection and therapeutic evaluation of solid cancers. However, the existing BLT reconstruction algorithms are not accurate enough for cavity cancer detection because of neglecting the void problem. Motivated by the ability of the hybrid radiosity-diffusion model (HRDM) in describing the light propagation in cavity organs, an HRDM-based BLT reconstruction algorithm was provided for the specific problem of cavity cancer detection. HRDM has been applied to optical tomography but is limited to simple and regular geometries because of the complexity in coupling the boundary between the scattering and void region. In the provided algorithm, HRDM was first applied to three-dimensional complicated and irregular geometries and then employed as the forward light transport model to describe the bioluminescent light propagation in tissues. Combining HRDM with the sparse reconstruction strategy, the cavity cancer cells labeled with bioluminescent probes can be more accurately reconstructed. Compared with the diffusion equation based reconstruction algorithm, the essentiality and superiority of the HRDM-based algorithm were demonstrated with simulation, phantom and animal studies. An in vivo gastric cancer-bearing nude mouse experiment was conducted, whose results revealed the ability and feasibility of the HRDM-based algorithm in the biomedical application of gastric cancer detection.

  7. Glioblastoma Therapy with Cytotoxic Mesenchymal Stromal Cells Optimized by Bioluminescence Imaging of Tumor and Therapeutic Cell Response

    PubMed Central

    Alieva, Maria; Bagó, Juli R.; Aguilar, Elisabet; Soler-Botija, Carolina; Vila, Olaia F.; Molet, Joan; Gambhir, Sanjiv S.; Rubio, Nuria; Blanco, Jerónimo

    2012-01-01

    Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 104 relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection. PMID:22529983

  8. Vision in lanternfish (Myctophidae): Adaptations for viewing bioluminescence in the deep-sea

    NASA Astrophysics Data System (ADS)

    Turner, J. R.; White, E. M.; Collins, M. A.; Partridge, J. C.; Douglas, R. H.

    2009-06-01

    The sensitivity hypothesis seeks to explain the correlation between the wavelength of visual pigment absorption maxima ( λmax) and habitat type in fish and other marine animals in terms of the maximisation of photoreceptor photon catch. In recent years its legitimacy has been called into question as studies have either not tested data against the output of a predictive model or are confounded by the wide phylogeny of species used. We have addressed these issues by focussing on the distribution of λmax values in one family of marine teleosts, the lanternfish (Myctophidae). Visual pigment extract spectrophotometry has shown that 54 myctophid species have a single pigment in their retinae with a λmax falling within the range 480-492 nm. A further 4 species contain two visual pigments in their retinae. The spectral distribution of these visual pigments seems relatively confined when compared to other mesopelagic fishes. Mathematical modelling based on the assumptions of the sensitivity hypothesis shows that, contrary to the belief that deep-sea fishes' visual pigments are shortwave shifted to maximise their sensitivity to downwelling sunlight, the visual pigments of myctophids instead seem better placed for the visualisation of bioluminescence. The predicted maximum visualisation distance of a blue/green bioluminescent point source by a myctophid was up to 30 m under ideal conditions. Two species ( Myctophum nitidulum and Bolinichthys longipes) have previously been shown to have longwave-shifted spectral sensitivities and we show that they could theoretically detect stomiid far-red bioluminescence from as far as ca. 7 m.

  9. Semi-automated Image Processing for Preclinical Bioluminescent Imaging.

    PubMed

    Slavine, Nikolai V; McColl, Roderick W

    Bioluminescent imaging is a valuable noninvasive technique for investigating tumor dynamics and specific biological molecular events in living animals to better understand the effects of human disease in animal models. The purpose of this study was to develop and test a strategy behind automated methods for bioluminescence image processing from the data acquisition to obtaining 3D images. In order to optimize this procedure a semi-automated image processing approach with multi-modality image handling environment was developed. To identify a bioluminescent source location and strength we used the light flux detected on the surface of the imaged object by CCD cameras. For phantom calibration tests and object surface reconstruction we used MLEM algorithm. For internal bioluminescent sources we used the diffusion approximation with balancing the internal and external intensities on the boundary of the media and then determined an initial order approximation for the photon fluence we subsequently applied a novel iterative deconvolution method to obtain the final reconstruction result. We find that the reconstruction techniques successfully used the depth-dependent light transport approach and semi-automated image processing to provide a realistic 3D model of the lung tumor. Our image processing software can optimize and decrease the time of the volumetric imaging and quantitative assessment. The data obtained from light phantom and lung mouse tumor images demonstrate the utility of the image reconstruction algorithms and semi-automated approach for bioluminescent image processing procedure. We suggest that the developed image processing approach can be applied to preclinical imaging studies: characteristics of tumor growth, identify metastases, and potentially determine the effectiveness of cancer treatment.

  10. Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

    PubMed Central

    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 (iv, ip, sc), 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