Decrease In Bioluminescence Research Articles

Quantification of human angiogenesis in immunodeficient mice using a photon counting-based method

Testing new antiangiogenic drugs for cancer treatment requires the use of animal models, since stromal cells and extracellular matrices mediate signals to endothelial cells that cannot be fully reproduced in vitro. Most methods used for analysis of antiangiogenic drugs in vivo utilized histologic examination of tissue specimens, which often requires large sample sizes to obtain reliable quantitative data. Furthermore, these assays rely on the analysis of murine vasculature that may not be correlated with the responses of human endothelial cells. Here, we engineered human blood vessels in immunodeficient mice with human endothelial cells expressing luciferase, demonstrated that these cells line functional blood vessels, and quantified angiogenesis over time using a photon counting-based method. In a proof-of-principle experiment with PTK/ZK, a small molecule inhibitor of vascular endothelial growth factor (VEGF) tyrosine kinase receptors, a strong correlation was observed between the decrease in bioluminescence (9.12-fold) in treated mice and the actual decrease in microvessel density (9.16-fold) measured after retrieval of the scaffolds and immunohistochemical staining of endothelial cells. The method described here allows for quantitative and noninvasive investigation into the effects of anti-cancer drugs on human angiogenesis in a murine host.

An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express α-fetoprotein

Antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In the present study, we first established a useful method to facilitate in vivo monitoring of attenuated S. typhimurium uptake using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. We found that the ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. We further examined the therapeutic efficacy of vaccination using attenuated S. typhimurium carrying the mouse alpha-fetoprotein (AFP) gene against a cancer line CT26-murine alpha-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. Attenuated S. typhimurium oral DNA vaccine was found to promote protective immunity against both CT26-mAFP and Hepa1-6 tumor cells growth. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development.

Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing

We have developed a highly sensitive biophotonic luciferase assay as an alternative to 51Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene ( fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo + target cells with CD8 + CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by 51Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.

Differences among antimicrobial properties of carrion beetle secretions reflect phylogeny and ecology.

Carrion beetles (Coleoptera: Silphidae) consist of two subfamilies in North America. Members of the Silphinae arrive at carcasses during the mid-stage of decay and their larvae feed on developing maggots, while members of the Nicrophorinae bury and tend carcasses upon which their developing larvae feed. The Nicrophorinae maintain the condition of the carcass by applying oral and anal secretions that reduce carcass decay apparently through bacterial inhibition, although quantification has not been made. We hypothesized that enzymes in the oral and anal secretions of the subfamily Nicrophorinae would inhibit bacterial growth, while secretions from the subfamily Silphinae would not. The secretions were assayed for inhibitory effects with a Microtox Analyzer that monitors the decrease in bioluminescence from the bacterium Vibrio fischerii. We found a significant difference of bioluminescence in the control compared to secretions of 8 out of 10 tested Nicrophorinae (with oral secretions being most active), while only anal secretions from Necrodes surinimensis of the Siphinae significantly reduced bacterial survival. These data follow the known phylogenic relationship in which Necrodes is the closest genus to the Nicrophorinae. The two species of Nicrophorinae, which did not show significant reductions in bacterial growth, differ ecologically from the others. Thus, the presence of antimicrobial compounds in most Nicrophorinae secretions, but not in most other Silphinae, represents an adaptation to preserve the buried carcass.

A novel continuous toxicity test system using a luminously modified freshwater bacterium

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3–8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox ® assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC 50-5 min values in an untreated raw wastewater sample (23.9 ± 12.8%) were the lowest, while those in an effluent sample (76.7 ± 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze–drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant

A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 ( lac:: luxCDABE), which constitutively produces bioluminescence. The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase. The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor. The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor. In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells. This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.

Statistical Optimization of Bioluminescence of Photobacterium posphoreum KCTC2852.

In this study, media optimization by statistically designed experiments stimulated an increase in the intensity and stability of bioluminescence of Photobacterium phosphoreum KCTC 2852 during continuous cultivation. The Plackett-Burman design method selected two components (NaCl, yeast extract) among nine components of the seawater complete (SC) medium. The response-surface design method calculated the optimum concentration of NaCl (31.1 g/l) and yeast extract (5.1 g/l). In the optimized medium, bioluminescence per cell increased twofold. During continuous cultivation in a stirred tank reactor, bioluminescence decreased rapidly in the SC medium, whereas the optimized medium could maintain bioluminescence for more than 80 h, resulting in an increased bioluminescence. Upon contact with 0.1 ppm Hg2+, a sharp decrease in bioluminescence was observed, which was reproducible in the measuring chamber. This shows the possibility that P. phosphoreum could be cultivated continuously with enhanced bioluminescence stability and that on-line water toxicity could be monitored continuously.

Statistical optimization of bioluminescence of Photobacterium phosphoreum KCTC2852

In this study, media optimization by statistically designed experiments stimulated an increase in the intensity and stability of bioluminescence of Photobacterium phosphoreum KCTC 2852 during continuous cultivation. The Plackett-Burman design method selected two components (NaCl, yeast extract) among nine components of the seawater complete (SC) medium. The response-surface design method calculated the optimum concentration of NaCl (31.1 g/ l) and yeast extract (5.1 g/ l). In the optimized medium, bioluminescence per cell increased twofold. During continuous cultivation in a stirred tank reactor, bioluminescence decreased rapidly in the SC medium, whereas the optimized medium could maintain bioluminescence for more than 80 h, resulting in an increased bioluminescence. Upon contact with 0.1 ppm Hg 2+, a sharp decrease in bioluminescence was observed, which was reproducible in the measuring chamber. This shows the possibility that P. phosphoreum could be cultivated continuously with enhanced bioluminescence stability and that on-line water toxicity could be monitored continuously.

Sediment quality assessment of flowing waters in South-West Germany using acute and chronic bioassays

Four bioassays were selected to examine sediment-elutriates of three flowing waters in the Saarland (South-West Germany). The aims of this study were the determination of the most sensitive bioassay for a sediment assessment within routine screening procedures and the comparison of a recently developed long-term bacterial assay with other commonly used and standardized bioassays. Sediment samples were elutriated with water and also with dimethyl sulphoxide in order to extract organic compounds. An expected accumulation of toxicants in the sediments was checked by additional examination of surface water at respective sampling sites, applying bioassays as well as chemical analysis methods. Whereas the sensitivity of the Daphnia magna assay was relatively low, the Scenedesmus subspicatus chlorophyll fluorescence test denoted the presence of high loads of nutrients at all sites. The results obtained using the Vibrio fischeri short-term bioassay suggested that an ecotoxicological risk can be excluded. The long-term luminescent bacteria assay is a further development of the short-term V. fischeri-assay. Prolonging the test duration from 30 min to 24 h should track down subacute toxicities, thus leading to a greater degree of sensitivity. However, the prolongation caused severe problems in respect of an accurate interpretation of the results. The long-term assay is not suited for routine deployment without major improvements. The extractable content of metals, pesticides, polychlorinated biphenyls and chlorinated hydrocarbons remained below their threshold values by several orders of magnitude. The use of dimethyl sulphoxide as solvent caused a decrease of bioluminescence in both bacterial assays. The inhibition was caused by toxic metabolic products of dimethyl sulphoxide formed by autochthonous bacteria from the sediment samples during the elutriation procedure. Thus, the use of dimethyl sulphoxide is not recommended for examinations of environmental samples when applying a luminescent bacteria assay.

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes.

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.

Sediment quality assessment of flowing waters in South-West Germany using acute and chronic bioassays

Abstract Four bioassays were selected to examine sediment-elutriates of three flowing waters in the Saarland (South-West Germany). The aims of this study were the determination of the most sensitive bioassay for a sediment assessment within routine screening procedures and the comparison of a recently developed long-term bacterial assay with other commonly used and standardized bioassays. Sediment samples were elutriated with water and also with dimethyl sulphoxide in order to extract organic compounds. An expected accumulation of toxicants in the sediments was checked by additional examination of surface water at respective sampling sites, applying bioassays as well as chemical analysis methods. Whereas the sensitivity of the Daphnia magna assay was relatively low, the Scenedesmus subspicatus chlorophyll fluorescence test denoted the presence of high loads of nutrients at all sites. The results obtained using the Vibrio fischeri short-term bioassay suggested that an ecotoxicological risk can be excluded. The long-term luminescent bacteria assay is a further development of the short-term V. fischeri-assay. Prolonging the test duration from 30 min to 24 h should track down subacute toxicities, thus leading to a greater degree of sensitivity. However, the prolongation caused severe problems in respect of an accurate interpretation of the results. The long-term assay is not suited for routine deployment without major improvements. The extractable content of metals, pesticides, polychlorinated biphenyls and chlorinated hydrocarbons remained below their threshold values by several orders of magnitude. The use of dimethyl sulphoxide as solvent caused a decrease of bioluminescence in both bacterial assays. The inhibition was caused by toxic metabolic products of dimethyl sulphoxide formed by autochthonous bacteria from the sediment samples during the elutriation procedure. Thus, the use of dimethyl sulphoxide is not recommended for examinations of environmental samples when applying a luminescent bacteria assay.

Effects of ultraviolet-B light and polyaromatic hydrocarbon exposure on sea urchin development and bacterial bioluminescence

Polycyclic aromatic hydrocarbons (PAHs) are relatively common contaminants of the Gulf of Mexico and may be activated to more toxic metabolites by ultraviolet-B (UV-B) light. A marine bacterial bioassay system ( Vibrio fischeri) which focused on the reduction of luciferase-mediated bioluminescence was utilized as a measure of toxicity to concurrent exposure to anthracene, benzo[a]pyrene, naphthalene, and phenanthrene and UV-B light (λ=280–320 nm). Inhibition of bacterial bioluminescence was observed following exposure to naphthalene (5 and 10 ppm) and phenanthrene (5 and 10 ppm) for 5 and 15 min. UV-B significantly ( p<0.05) decreased the average bacterial bioluminescence in all treatments by 23.8%, as compared to control. No significant decrease in bioluminescence was observed with anthracene and benzo[a]pyrene at concentrations up to 24 and 1.6 ppm, respectively. Anthracene and benzo[a]pyrene are relatively insoluble in water (<45 ppb); therefore, the bioavailability of the two PAHs may significantly influence the exposure and effects of anthracene and benzo[a]pyrene to V. fischeri. Results of bacterial bioluminescence experiments were compared to a sea urchin ( Lytechinus variegatus) development assay. Development of sea urchin embryos to the four- and 32-cell stage was utilized as a measure of toxicity to the combined effects of UV-B light and the PAHs, benzo[a]pyrene and phenanthrene. Developing sea urchins demonstrated a significant ( p<0.05) dose-dependent decrease in growth rate following exposure to benzo[a]pyrene (1, 5, 50, and 100 ppb) and phenanthrene (1, 5, 50, and 100 ppb), with UV-B light (5.8 μW/cm 2). Interaction analysis demonstrates that concurrent exposure to UV-B and PAHs results in additive toxicological effects in both marine organisms tested. Results of the present study suggest that environmental conditions present in the Gulf of Mexico, including both UV-B light and PAH exposure, have the potential to cause adverse toxicological effects to marine organisms.

Bioluminescent Reporter Bacterium for Toxicity Monitoring in Biological Wastewater Treatment Systems

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 (0.01 to 10 000 mg/L), 2,4‐dinitrophenol (0.01 to 1 000 mg/L), and hydroquinone (0.01 to 10 000 mg/L) 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 10 000 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.

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[ a]pyrene were produced using 50 mM hydroxpropyl-β-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[ a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using 14C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons.

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[a]pyrene were produced using 50 mM hydroxpropyl-beta-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using 14C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.

Use of a lux‐modified bacterial biosensor to identify constraints to bioremediation of btex‐contaminated sites

AbstractSediment and groundwater samples obtained from a benzene, toluene, ethylbenzene, and xylene (BTEX)‐contaminated site were screened, using a bioluminescence‐based (lux gene‐marked) bacterial biosensor, to identify constraints to site remediation. Through a series of sample manipulations and linked biosensor responses, constraints to BTEX remediation such as adverse pH, presence of nonvolatile organic contaminants, and the presence of heavy metals were investigated. Conventional chemical analysis (gas chromatography, inductively coupled plasma mass spectroscopy) was used to confirm the reliable performance of the biosensor and to identify its potential contribution to site management to ensure effective remediation. The toxicity results of the biosensor were expressed in % maximum bioluminescence calculated against a blank of double deionized water with pH adjusted to 5.5. Untreated samples caused reductions in percentage bioluminescence from 10 to 95%. Water sample W1, containing a total BTEX concentration of 30,595 μg/L caused the highest decrease in bioluminescence (5.78%). This toxicity was significantly reduced after elimination of volatile organic compounds (VOCs) (42.01%) and bioluminescence was further increased to 87.80% after removal of total organic matter. This suggested that VOCs were not the only toxic organopollutants present. Water samples giving high values of bioluminescence contained BTEX concentrations of 17 to 31 μg/L.

Electromagnetic field effect on luminescent bacteria

The effects of electromagnetic fields (EMFs) with frequencies varying from 36 to 55 GHz on the bioluminescent activity of bacteria were investigated. EMFs resulted in a decrease of bioluminescence which depended on frequency. The time of adaptation of cells to the electromagnetic field was longer than the intrinsic temporal parameters of the bioluminescent system. The effect had a nonthermal nature. It was found that disturbances of the geomagnetic field (magnetic storms) resulted in an increase of bioluminescence.