Sample records for chip-scale bioassays based

  1. Chip-Scale Bioassays Based on Surface-Enhanced Raman Scattering: Fundamentals and Applications

    Energy Technology Data Exchange (ETDEWEB)

    Hye-Young Park


    This work explores the development and application of chip-scale bioassays based on surface-enhanced Raman scattering (SERS) for high throughput and high sensitivity analysis of biomolecules. The size effect of gold nanoparticles on the intensity of SERS is first presented. A sandwich immunoassay was performed using Raman-labeled immunogold nanoparticles with various sizes. The SERS responses were correlated to particle densities, which were obtained by atomic force microscopy (AFM). The response of individual particles was also investigated using Raman-microscope and an array of gold islands on a silicon substrate. The location and the size of individual particles were mapped using AFM. The next study describes a low-level detection of Escherichia coli 0157:H7 and simulants of biological warfare agents in a sandwich immunoassay format using SERS labels, which have been termed Extrinsic Raman labels (ERLs). A new ERL scheme based on a mixed monolayer is also introduced. The mixed monolayer ERLs were created by covering the gold nanoparticles with a mixture of two thiolates, one thiolate for covalently binding antibody to the particle and the other thiolate for producing a strong Raman signal. An assay platform based on mixed self-assembled monolayers (SAMs) on gold is then presented. The mixed SAMs were prepared from dithiobis(succinimidyl undecanoate) (DSU) to covalently bind antibodies on gold substrate and oligo(ethylene glycol)-terminated thiol to prevent nonspecific adsorption of antibodies. After the mixed SAMs surfaces, formed from various mole fraction of DSU were incubated with antibodies, AFM was used to image individual antibodies on the surface. The final study presents a collaborative work on the single molecule adsorption of YOYO-I labeled {lambda}-DNA at compositionally patterned SAMs using total internal reflection fluorescence microscopy. The role of solution pH, {lambda}-DNA concentration, and domain size was investigated. This work also revealed

  2. Chip-Scale Bioassays Based on Surface-Enhanced Raman Scattering: Fundamentals and Applications

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hye-Young [Iowa State Univ., Ames, IA (United States)


    This work explores the development and application of chip-scale bioassays based on surface-enhanced Raman scattering (SERS) for high throughput and high sensitivity analysis of biomolecules. The size effect of gold nanoparticles on the intensity of SERS is first presented. A sandwich immunoassay was performed using Raman-labeled immunogold nanoparticles with various sizes. The SERS responses were correlated to particle densities, which were obtained by atomic force microscopy (AFM). The response of individual particles was also investigated using Raman-microscope and an array of gold islands on a silicon substrate. The location and the size of individual particles were mapped using AFM. The next study describes a low-level detection of Escherichia coli 0157:H7 and simulants of biological warfare agents in a sandwich immunoassay format using SERS labels, which have been termed Extrinsic Raman labels (ERLs). A new ERL scheme based on a mixed monolayer is also introduced. The mixed monolayer ERLs were created by covering the gold nanoparticles with a mixture of two thiolates, one thiolate for covalently binding antibody to the particle and the other thiolate for producing a strong Raman signal. An assay platform based on mixed self-assembled monolayers (SAMs) on gold is then presented. The mixed SAMs were prepared from dithiobis(succinimidyl undecanoate) (DSU) to covalently bind antibodies on gold substrate and oligo(ethylene glycol)-terminated thiol to prevent nonspecific adsorption of antibodies. After the mixed SAMs surfaces, formed from various mole fraction of DSU were incubated with antibodies, AFM was used to image individual antibodies on the surface. The final study presents a collaborative work on the single molecule adsorption of YOYO-I labeled {lambda}-DNA at compositionally patterned SAMs using total internal reflection fluorescence microscopy. The role of solution pH, {lambda}-DNA concentration, and domain size was investigated. This work also revealed

  3. Bioassays Based on Molecular Nanomechanics

    Directory of Open Access Journals (Sweden)

    Arun Majumdar


    Full Text Available Recent experiments have shown that when specific biomolecular interactions are confined to one surface of a microcantilever beam, changes in intermolecular nanomechanical forces provide sufficient differential torque to bend the cantilever beam. This has been used to detect single base pair mismatches during DNA hybridization, as well as prostate specific antigen (PSA at concentrations and conditions that are clinically relevant for prostate cancer diagnosis. Since cantilever motion originates from free energy change induced by specific biomolecular binding, this technique is now offering a common platform for label-free quantitative analysis of protein-protein binding, DNA hybridization DNA-protein interactions, and in general receptor-ligand interactions. Current work is focused on developing “universal microarrays” of microcantilever beams for high-throughput multiplexed bioassays.

  4. Nanoparticle-Based Biosensors and Bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guodong; Wang, Jun; Lin, Yuehe; Wang, Joseph


    In this book chapter, we review the recent advances in nanoparticles based bioassay. The nanoparticles include quantum dots, silica nanoparticles and apoferritin nanoparticles. The new nanoparticles-based labels hold great promise for multiplex protein and DNA detection and for enhancing the sensitivity of other bioassays.

  5. Chip scale mechanical spectrum analyzers based on high quality factor overmoded bulk acouslic wave resonators

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, R. H., III


    The goal of this project was to develop high frequency quality factor (fQ) product acoustic resonators matched to a standard RF impedance of 50 {Omega} using overmoded bulk acoustic wave (BAW) resonators. These resonators are intended to serve as filters in a chip scale mechanical RF spectrum analyzer. Under this program different BAW resonator designs and materials were studied theoretically and experimentally. The effort resulted in a 3 GHz, 50 {Omega}, sapphire overmoded BAW with a fQ product of 8 x 10{sup 13}, among the highest values ever reported for an acoustic resonator.

  6. Large-field-of-view Chip-scale Talbot-grid-based Fluorescence Microscopy

    CERN Document Server

    Pang, Shuo; Kato, Mihoko; Sternberg, Paul W; Yang, Changhuei


    The fluorescence microscope is one of the most important tools in modern clinical diagnosis and biological science. However, its expense, size and limited field-of-view (FOV) are becoming bottlenecks in key applications such as large-scale phenotyping and low-resource-setting diagnostics. Here we report a low-cost, compact chip-scale fluorescence-imaging platform, termed the Fluorescence Talbot Microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly-scalable FOV. The FTM prototype has a resolution of 1.2 microns and an FOV of 3.9 mm x 3.5 mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and HEK cells expressing green fluorescent protein.

  7. Phonon-based scalable platform for chip-scale quantum computing (United States)

    Reinke, Charles M.; El-Kady, Ihab


    We present a scalable phonon-based quantum computer on a phononic crystal platform. Practical schemes involve selective placement of a single acceptor atom in the peak of the strain field in a high-Q phononic crystal cavity that enables coupling of the phonon modes to the energy levels of the atom. We show theoretical optimization of the cavity design and coupling waveguide, along with estimated performance figures of the coupled system. A qubit can be created by entangling a phonon at the resonance frequency of the cavity with the atom states. Qubits based on this half-sound, half-matter quasi-particle, called a phoniton, may outcompete other quantum architectures in terms of combined emission rate, coherence lifetime, and fabrication demands.

  8. Microfabricated cells for chip-scale atomic clock based on coherent population trapping: Fabrication and investigation

    Directory of Open Access Journals (Sweden)

    S.V. Ermak


    Full Text Available A universal method for fabrication of miniature cells for frequency standards and quantum magnetometers containing 87Rb atoms in the atmosphere of inert gas neon based on integrated technologies is considered. The results of experimental studies of coherent population trapping signals observed for a series of cells which provided recovery of vapors of an alkali metal from the rubidium dichromate salt with the help of laser radiation are presented. The coherent population trapping signals with a typical linewidth of 2–3 kHz and a signal-to-noise ratio of 1500 in the 1-Hz bandwidth were observed, which allows one to provide a relative frequency stability of atomic clock of 10−11 at 100 s.

  9. Fabrication method for chip-scale-vacuum-packages based on a chip-to-wafer-process (United States)

    Bauer, J.; Weiler, D.; Ruß, M.; Heß, J.; Yang, P.; Voß, J.; Arnold, N.,; Vogt, H.


    This paper introduces a simple vacuum packaging method which is based on a Chip-to-Wafer process. The MEMS-device is provided with an electroplated solder frame. A Si-lid with the same solder frame is mounted on each die of the wafer using a flip chip process. The same materials for lid and substrate are used in order to reduce the mechanical stress due to the same thermal coefficients of expansion. The resulting cavity between die and lid can be evacuated and hermetically sealed with an eutectic soldering process. The feasibility of the method is demonstrated with an infrared focal plane array (IR-FPA). In this case, the Si-lid acts as an optical window and contains an anti reflective layer for the 8-14 μm wavelength area on both sides. The long-term vacuum stability is supported by a getter film inside the package. This method simplifies the sawing process and has the additional cost benefit that it is possible to package only known good dies.

  10. Nanomaterial-Based Electrochemical Biosensors and Bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guodong; Mao, Xun; Gurung, Anant; Baloda, Meenu; Lin, Yuehe; He, Yuqing


    This book chapter summarizes the recent advance in nanomaterials for electrochemical biosensors and bioassays. Biofunctionalization of nanomaterials for biosensors fabrication and their biomedical applications are discussed.

  11. Cell-based bioassays in microfluidic systems (United States)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.


    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  12. [Investigation on pattern and methods of quality control for Chinese materia medica based on dao-di herbs and bioassay - bioassay for Coptis chinensis]. (United States)

    Yan, Dan; Xiao, Xiao-he


    Establishment of bioassay methods is the technical issues to be faced with in the bioassay of Chinese materia medica. Taking the bioassay of Coptis chinensis Franch. as an example, the establishment process and application of the bioassay methods (including bio-potency and bio-activity fingerprint) were explained from the aspects of methodology, principle of selection, experimental design, method confirmation and data analysis. The common technologies were extracted and formed with the above aspects, so as to provide technical support for constructing pattern and method of the quality control for Chinese materia medica based on the dao-di herbs and bioassay.

  13. Chip-scale spacecraft swarms: Dynamics, control, and exploration (United States)

    Weis, Lorraine

    Chip-scale spacecraft (chipsats) swarms will open new avenues for space exploration, both near Earth and in interplanetary space. The ability to create distributed sensor networks through swarms of low-cost, low-mass spacecraft shall enable the exploration of asteroids, icy moons, and the Earths magnetosphere become more feasible. This research develops new techniques for analyzing swarm dynamics, both in the limited case of the Kepler problem, and in general gravity environments, investigates several techniques for providing chipsat propulsion, and develops possible mission strategies. This work applies the Kustaanheimo-Stiefel (KS) transformation to the stochastic exploration presented by chipsat swarms. The contributions towards understanding swarm dynamics include analytical and numerical study of swarms in the purely Kepler problem as well as in general potential fields. A study of swarm evolution near an asteroid provides an example of the richness of behaviors that can be provided by chip-scale spacecraft swarms. Swarm actuation can be achieved through a number of means. This research presents a novel attitude control and propulsion system for chipsat swarms near Earth using a mutliple electrodynamic tethers. A numerical study of tether configurations for the greatest control authority is also presented. In addition, active solar sails are evaluated for swarm actuation beyond Earth, and a visualization of available control authority is presented. An example mission of swarm deployment near the Earth-Moon Lagrange point highlights the utility of swarm-based exploration. The candidate mission shows that a swarm with minimal actuation and a simple control scheme might provide distributed sensors in the region for a year or more, or dissipate quickly if uncontrolled. Such a chip-spacecraft mission would be a valuable precursor to further space development in these regions.

  14. A fluorescence-based bioassay for antibacterials and its application in screening natural product extracts

    NARCIS (Netherlands)

    Michels, Katharina; Heinke, Ramona; Schöne, Pia; Kuipers, Oscar P; Arnold, Norbert; Wessjohann, Ludger A


    The reliable assessment of the biological activity of a minor component embedded in a complex matrix of several hundred compounds is a difficult but common task in the search for natural product-based antibiotics, for example, by bioassay-guided fractionation. To quantify the antibiotic properties,

  15. Does co-extracted dissolved organic carbon cause artefacts in cell-based bioassays? (United States)

    Neale, Peta A; Escher, Beate I


    Bioanalytical tools are increasingly being employed for water quality monitoring, with applications including samples that are rich in natural organic matter (or dissolved organic carbon, DOC), such as wastewater. While issues associated with co-extracted DOC have been identified for chemical analysis and for bioassays with isolated enzymes, little is known about its effect on cell-based bioassays. Using mixture experiments as diagnostic tools, this study aims to assess whether different molecular weight fractions of wastewater-derived DOC adversely affect cell-based bioassays, specifically the bioluminescence inhibition test with the bacteria Vibrio fischeri, the combined algae assay with Pseudokirchneriella subcapitata and the human cell line AREc32 assay for oxidative stress. DOC did not cause suppressive effects in mixtures with reference compounds. Binary mixtures further indicated that co-extracted DOC did not disturb cell-based bioassays, while slight deviations from toxicity predictions for low molecular weight fractions may be partially due to the availability of natural components to V. fischeri, in addition to organic micropollutants.

  16. Paper-based chromatic toxicity bioassay by analysis of bacterial ferricyanide reduction. (United States)

    Pujol-Vila, F; Vigués, N; Guerrero-Navarro, A; Jiménez, S; Gómez, D; Fernández, M; Bori, J; Vallès, B; Riva, M C; Muñoz-Berbel, X; Mas, J


    Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox(®), rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at -20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC50) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation

  17. Development of bacteria-based bioassays for arsenic detection in natural waters

    Energy Technology Data Exchange (ETDEWEB)

    Diesel, Elizabeth; Schreiber, Madeline [Virginia Tech, Department of Geosciences, Blacksburg, VA (United States); Meer, Jan Roelof van der [University of Lausanne, Department of Fundamental Microbiology, Lausanne (Switzerland)


    Arsenic contamination of natural waters is a worldwide concern, as the drinking water supplies for large populations can have high concentrations of arsenic. Traditional techniques to detect arsenic in natural water samples can be costly and time-consuming; therefore, robust and inexpensive methods to detect arsenic in water are highly desirable. Additionally, methods for detecting arsenic in the field have been greatly sought after. This article focuses on the use of bacteria-based assays as an emerging method that is both robust and inexpensive for the detection of arsenic in groundwater both in the field and in the laboratory. The arsenic detection elements in bacteria-based bioassays are biosensor-reporter strains; genetically modified strains of, e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Rhodopseudomonas palustris. In response to the presence of arsenic, such bacteria produce a reporter protein, the amount or activity of which is measured in the bioassay. Some of these bacterial biosensor-reporters have been successfully utilized for comparative in-field analyses through the use of simple solution-based assays, but future methods may concentrate on miniaturization using fiberoptics or microfluidics platforms. Additionally, there are other potential emerging bioassays for the detection of arsenic in natural waters including nematodes and clams. (orig.)

  18. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. (United States)

    Sonneveld, Edwin; Jansen, Hendrina J; Riteco, Jacoba A C; Brouwer, Abraham; van der Burg, Bart


    We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERalpha (estrogen receptor alpha) CALUX (Chemically Activated LUciferase eXpression), respectively. Both bioassays are member of a panel of CALUX reporter cell lines derived from the human U2-OS osteosarcoma cell line, all using highly selective reporter constructs based with a basal promoter element linked to multimerized response elements, allowing efficient and specific measurement of compounds interfering with androgen, estrogen, progesterone, and glucocorticoid receptors. The AR CALUX bioassay contains the human androgen receptor and a luciferase reporter construct containing three androgen-responsive elements coupled to a minimal TATA promoter. This cell line was characterized by its stable expression of AR protein, its highly selective response to low levels of different natural and synthetic androgens, and its insignificant response to other nuclear hormone receptor ligands such as estrogens, progestins, and glucocorticoids. The EC50 of dihydrotestosterone (DHT) was found to be 0.13 nM, consistent with the high affinity of this ligand to the human AR. Flutamide, cyproterone acetate, and the environmental contaminants vinclozolin, DDT, methoxychlor, its metabolite HPTE, and penta-BFR showed clear antagonistic activity in the AR CALUX bioassay, competitively inhibiting DHT-mediated transactivation. The established AR CALUX bioassay proved to excel in terms of easy cell line maintenance, high fold induction range (typical 30 times over solvent control), low minimal detection limit (3.6 pM), and high androgen selectivity. Potential applications such as testing the androgenic or estrogenic activity of pure chemicals and pharmaceuticals and complex mixtures (environmental, food, feed, and clinical) are discussed.

  19. Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics. (United States)

    Pujol-Vila, F; Vigués, N; Díaz-González, M; Muñoz-Berbel, X; Mas, J


    Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.

  20. Bioassay Laboratory (United States)

    Federal Laboratory Consortium — The Bioassay Laboratory is an accredited laboratory capable of conducting standardized and innovative environmental testing in the area of aquatic ecotoxicology. The...

  1. [Investigation on pattern of quality control for Chinese materia medica based on famous-region drug and bioassay--the work reference]. (United States)

    Yan, Dan; Xiao, Xiaohe


    Selection and standardization of the work reference are the technical issues to be faced with in the bioassay of Chinese materia medica. Taking the bioassay of Coptis chinensis. as an example, the manufacture process of the famous-region drugs extraction was explained from the aspects of original identification, routine examination, component analysis and bioassay. The common technologies were extracted, and the selection and standardization procedures of the work reference for the bioassay of Chinese materia medica were drawn up, so as to provide technical support for constructing a new mode and method of the quality control of Chinese materia medica based on the famous-region drugs and bioassay.

  2. Biotesting of radioactively contaminated forest soils using barley-based bioassay (United States)

    Mel’nikova, T. V.; Polyakova, L. P.; Oudalova, A. A.


    Findings from radioactivity and phytotoxicity study are presented for soils from nine study-sites of the Klintsovsky Forestry located in the Bryansk region that were radioactively contaminated after the Chernobyl accident. According to the bioassay based on barley as test-species, stimulating effect of the soils analyzed is revealed for biological indexes of the length of barley roots and sprouts. From data on 137Cs specific activities in soils and plant biomass, the migration potential of radionuclide in the "soil-plant" system is assessed as a transfer factor. With correlation analysis, an impact of 137Cs in soil on the biological characteristics of barley is estimated.

  3. Application of a lux-based bioassay to assess soil toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Paton, G.I. [Macaulay Land Use Research Inst., Aberdeen (United Kingdom)]|[Univ. of Aberdeen (United Kingdom); Campbell, C.D. [Macaulay Land Use Research Inst., Aberdeen (United Kingdom); Rattray, E.A.S.; Glover, L.A.; Killham, K. [Univ. of Aberdeen (United Kingdom)


    The expression of prokaryotic bioluminescence is linked with cell metabolism and accordingly bioassays have been developed using naturally bioluminescent bacteria to assess ecotoxicity. Advances in biotechnology have allowed the isolation of the lux genes (responsible for bioluminescence) from marine organisms and their insertion into terrestrial bacteria. This has enabled the use of ecologically relevant bacteria to assess toxicity by measuring bioluminescence response in the presence of toxins. The lux genes were inserted into Pseudomonas fluorescens and Rhizobium leguminosarum biovar trifolii as a multi-copy plasmid and also integrated into the chromosome. It was found that in aqueous solutions the plasmid constructs were more sensitive than the chromosomal constructs to a range of toxins. The order of toxicity for Ps. fluorescens was Zn = Cu > Cd > Ni > Cr > DCP and for R. trifolii Zn > Cu > Cd > DCP > Cr. The lux based bioassays were more reproducible and sensitive than ATP and dehydrogenase assays and offered greater sensitivity than Photobacterium phosphoreum assays to assess toxicity of inorganic pollutants. Extracts from 4 soil types were spiked with a range of toxins and when EC{sub 50} values were determined it was shown that toxicity was related to soil characteristics. This enabled the assay to be used to assess the Lee Valley soil experiment which represents an important international study of the effect of the application of contaminated sewage to land. High metal application rates had been shown to have serious implications for soil ecology. Chemical analysis, carried out 26 years after sewage addition confirmed that soil extracts still had increased metal concentrations. The lux-based bioassays, which proved to be rapid, reproducible and sensitive confirmed that the metals were still biologically available and hence toxic.

  4. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release. (United States)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen; Hansen, Anders H; Mollenhauer, Jan; Mouritsen, Ole G


    The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

  5. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. (United States)

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger


    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  6. Single-core magnetic markers in rotating magnetic field based homogeneous bioassays and the law of mass action

    Energy Technology Data Exchange (ETDEWEB)

    Dieckhoff, Jan, E-mail: [Institut fuer Elektrische Messtechnik und Grundlagen der Elektrotechnik, TU Braunschweig, Braunschweig (Germany); Schrittwieser, Stefan; Schotter, Joerg [Molecular Diagnostics, AIT Austrian Institute of Technology, Vienna (Austria); Remmer, Hilke; Schilling, Meinhard; Ludwig, Frank [Institut fuer Elektrische Messtechnik und Grundlagen der Elektrotechnik, TU Braunschweig, Braunschweig (Germany)


    In this work, we report on the effect of the magnetic nanoparticle (MNP) concentration on the quantitative detection of proteins in solution with a rotating magnetic field (RMF) based homogeneous bioassay. Here, the phase lag between 30 nm iron oxide single-core particles and the RMF is analyzed with a fluxgate-based measurement system. As a test analyte anti-human IgG is applied which binds to the protein G functionalized MNP shell and causes a change of the phase lag. The measured phase lag changes for a fixed MNP and a varying analyte concentration are modeled with logistic functions. A change of the MNP concentration results in a nonlinear shift of the logistic function with the analyte concentration. This effect results from the law of mass action. Furthermore, the bioassay results are used to determine the association constant of the binding reaction. - Highlights: • A rotating magnetic field based homogeneous bioassay concept was presented. • Here, single-core iron oxide nanoparticles are applied as markers. • The impact of the particle concentration on the bioassay results is investigated. • The relation between particle concentration and bioassay sensitivity is nonlinear. • This finding can be reasonably explained by the law of mass action.

  7. Toxicity of some bis Mannich bases and corresponding piperidinols in the brine shrimp (Artemia salina) bioassay. (United States)

    Gul, H Inci; Gul, Mustafa; Erciyas, Ercin


    Some acetophenone-derived bis Mannich bases were synthesized: bis[beta-benzoylethyl]ethylamine hydrochloride (IIa), bis[beta-(p-methylbenzoyl)ethyl]ethylamine hydrochloride (IIb), bis[beta-(p-chlorobenzoyl)ethyl]ethy- lamine hydrochloride (IId), bis[(2-thienylcarbonyl)ethyl]ethylamine hydrochloride (IIe); some corresponding piperidinol derivatives: 3-benzoyl-1-ethyl-4-phenyl-4-piperidinol hydrochloride (IIIa), 1-ethyl-3-(p-methyl- benzoyl)-4-(p-methylphenyl)-4-piperidinol hydrochloride (IIIb), 1-ethyl-3-(p-methoxybenzoyl)-4-(p-methoxy- phenyl)-4-piperidinol hydrochloride (IIIc), 1-ethyl-3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-4-piperidinol hydrochloride (IIId), 1-ethyl-4-(2-thienyl)-3-(2-thienylcarbonyl)-4-piperidinol hydrochloride (IIIe); and some representative quaternary piperidinols: 3-benzoyl-1-ethyl-4-hydroxy-1-methyl-4-phenylpiperidinium iodide (IIIf), 1-ethyl-4-hydroxy-1-methyl-3-(p-methylbenzoyl)-4-(p-methylphenyl)piperidinium iodide (IIIg). Toxicity was tested by the brine shrimp bioassay as an intermediate test before further in vivo animal experiments. Piperidine derivatives were found to be more potent than bis Mannich bases. Quaternary piperidine derivatives IIIf and IIIg and also non-quaternary piperidine derivatives IIIb, IIIe, IIIc and IIId were more toxic than 5-fluorouracil in brine shrimp bioassay. Except for IIe, bis Mannich bases were not effective. Quaternization and conversion of bis Mannich bases to corresponding piperidines improved the toxicity. The lipid solubility of the compounds may not affect the toxicity. From these findings the quaternary piperidine derivatives IIIf and IIIg could be used in further drug development and also for in vivo experiments.

  8. Chip-scale cavity optomechanics in lithium niobate

    CERN Document Server

    Jiang, Wei C


    We develop a chip-scale cavity optomechanical system in single-crystal lithium niobate that exhibits high optical quality factors and a large frequency-quality product as high as $3.6\\times 10^{12}$ Hz at room temperature and atmosphere. The excellent optical and mechanical properties together with the strong optomechanical coupling allow us to efficiently excite the coherent regenerative optomechanical oscillation operating at 375.8 MHz with a threshold power of 174 ${\\rm \\mu W}$ in the air. The demonstrated lithium niobate optomechanical device enables great potential for achieving electro-optic-mechanical hybrid systems for broad applications in sensing, metrology, and quantum physics.

  9. Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti). (United States)

    Stell, F M; Roe, R M; Arellano, C; Kennedy, L; Thornton, H; Saavedra-Rodriguez, K; Wesson, D M; Black, W C; Apperson, C S


    Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar-insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose-permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose-permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar-insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field-collected strains was characterized by probit analysis of dosage-response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage-response patterns were similar, indicating that the sugar-insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.

  10. Effect-based trigger values for in vitro bioassays: Reading across from existing water quality guideline values. (United States)

    Escher, Beate I; Neale, Peta A; Leusch, Frederic D L


    Cell-based bioassays are becoming increasingly popular in water quality assessment. The new generations of reporter-gene assays are very sensitive and effects are often detected in very clean water types such as drinking water and recycled water. For monitoring applications it is therefore imperative to derive trigger values that differentiate between acceptable and unacceptable effect levels. In this proof-of-concept paper, we propose a statistical method to read directly across from chemical guideline values to trigger values without the need to perform in vitro to in vivo extrapolations. The derivation is based on matching effect concentrations with existing chemical guideline values and filtering out appropriate chemicals that are responsive in the given bioassays at concentrations in the range of the guideline values. To account for the mixture effects of many chemicals acting together in a complex water sample, we propose bioanalytical equivalents that integrate the effects of groups of chemicals with the same mode of action that act in a concentration-additive manner. Statistical distribution methods are proposed to derive a specific effect-based trigger bioanalytical equivalent concentration (EBT-BEQ) for each bioassay of environmental interest that targets receptor-mediated toxicity. Even bioassays that are indicative of the same mode of action have slightly different numeric trigger values due to differences in their inherent sensitivity. The algorithm was applied to 18 cell-based bioassays and 11 provisional effect-based trigger bioanalytical equivalents were derived as an illustrative example using the 349 chemical guideline values protective for human health of the Australian Guidelines for Water Recycling. We illustrate the applicability using the example of a diverse set of water samples including recycled water. Most recycled water samples were compliant with the proposed triggers while wastewater effluent would not have been compliant with a few

  11. A fluorescence-based bioassay for antibacterials and its application in screening natural product extracts. (United States)

    Michels, Katharina; Heinke, Ramona; Schöne, Pia; Kuipers, Oscar P; Arnold, Norbert; Wessjohann, Ludger A


    The reliable assessment of the biological activity of a minor component embedded in a complex matrix of several hundred compounds is a difficult but common task in the search for natural product-based antibiotics, for example, by bioassay-guided fractionation. To quantify the antibiotic properties, it is necessary to assess the cell viability. Direct measurements use CFU counts, OD measurements or detection via fluorescent or reducible dyes. However, natural extracts often already possess intrinsic dye, fluorescent, reducing or protein denaturing properties, or they contain insoluble compounds or general protein-binding (tanning) polyphenols as disturbing features, while at the same time very little of the selective antibiotic sought after is present. A promising alternative is provided by intrinsically produced bright fluorescent proteins. In this paper, a rapid, robust and concentration-dependent assay for screening antibiotics with genetically modified mutants of Bacillus subtilis 168 (PabrB-iyfp) is presented. The Gram-positive bacteria exhibit a native fluorescence during their exponential growth phase due to the expression of improved yellow fluorescent protein. To demonstrate the applicability in the field of natural product research, several compounds and extracts were screened for antibacterial activity, with an emphasis on those from the fungal genus Hygrophorus (waxy caps).

  12. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays (United States)

    Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen


    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

  13. Toxicological profiling of sediments with in vitro mechanisms-based bioassays for endocrine disruption

    NARCIS (Netherlands)

    Houtman, C.J.; Cenijn, P.H.; Hamers, T.; Lamoree, M.H.; Legler, J.; Murk, A.J.; Brouwer, A.


    In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in v

  14. A new mass spectrometry based bioassay for the direct assessment of hyaluronidase activity and inhibition. (United States)

    Britton, Emily R; Ibberson, Carolyn B; Horswill, Alexander R; Cech, Nadja B


    The development of drug resistance by bacterial pathogens is a growing threat. Drug resistant infections have high morbidity and mortality rates, and treatment of these infections is a major burden on the health care system. One potential strategy to prevent the development of drug resistance would be the application of therapeutic strategies that target bacterial virulence. Hyaluronidase is virulence factor that plays a role in the ability of Gram-positive bacteria such as Staphyloccus aureus and Streptococcus agalactiae to spread in tissue. As such, this enzyme could be a target for the development of future anti-virulence therapies. To facilitate the identification of hyaluronidase inhibitors, quantitative and reproducible assays of hyaluronidase activity are required. In the present study, we developed a new mass spectrometry based bioassay for this purpose. This assay directly measures the quantity of a degradation product (3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine) produced by the hyaluronidase enzyme. Validation parameters for the new assay are as follows: repeatability, <7%; intermediate precision, <10%; range, 0.78-50 μM; limit of detection, 0.29 μM; and limit of quantification, 0.78 μM. Using the new assay, the IC50 value for a published inhibitor of S. agalactiae hyaluronidase, ascorbic acyl 6-palmitate, was 8.0±1.0 μM. We also identified a new hyaluronidase inhibitor, n-cyclohexanecarbonylpentadecylamine, with an IC50 of 30.4±9.8 μM. In conclusion, we describe a new, direct, and reproducible method for assessing hyaluronidase activity using mass spectrometry that can facilitate the discovery of inhibitors.

  15. Imaging and identification of waterborne parasites using a chip-scale microscope.

    Directory of Open Access Journals (Sweden)

    Seung Ah Lee

    Full Text Available We demonstrate a compact portable imaging system for the detection of waterborne parasites in resource-limited settings. The previously demonstrated sub-pixel sweeping microscopy (SPSM technique is a lens-less imaging scheme that can achieve high-resolution (<1 µm bright-field imaging over a large field-of-view (5.7 mm×4.3 mm. A chip-scale microscope system, based on the SPSM technique, can be used for automated and high-throughput imaging of protozoan parasite cysts for the effective diagnosis of waterborne enteric parasite infection. We successfully imaged and identified three major types of enteric parasite cysts, Giardia, Cryptosporidium, and Entamoeba, which can be found in fecal samples from infected patients. We believe that this compact imaging system can serve well as a diagnostic device in challenging environments, such as rural settings or emergency outbreaks.

  16. Development and characterization of a green fluorescent protein-based rat cell bioassay system for detection of AH receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Bin; Denison, M. [California Univ., Davis, CA (United States). Dept. of Environmental Toxicology


    Proper epidemiological, risk assessment and exposure analysis of TCDD and related HAHs requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). While high-resolution instrumental analysis techniques are established for these chemicals, these procedures are very costly, time-consuming and are impractical for large scale sampling studies. Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take the advantage of the ability of these chemicals to activate one or more aspects of the AhR-dependent mechanism of action. One of the most sensitive bioassay systems developed to date is the so-called CALUX (Chemically Activated Luciferase Expression) assay, which is based on novel recombinant cell lines that contain a stably transfected dioxin (AhR)-responsive firefly luciferase gene. Treatment of these cells with TCDD and related HAHs and polycyclic aromatic hydrocarbons (PAHs), as well as other AhR ligands, results in induction of reporter gene expression in a time-, dose-, AhR-, and chemical-specific manner. The level of reporter gene expression correlates with the total concentration of the TCDD-like AhR inducers (agonists) present in the sample. Although the firefly luciferase reporter gene contributes to the high degree of sensitivity of the assay, it also has limitations with respect to our need for a rapid and inexpensive bioassay for high-throughput screening analysis. Accordingly, we previously developed a stably transfected murine cell line containing an AhRresponsive enhanced green fluorescent protein (EGFP) reporter gene. This cell line provided us with a high-throughput cell bioassay system for identification and characterization of AhR agonists and antagonists. Here we have extended these studies and describe the development, optimization, and

  17. Chip-scale Photonic Devices for Light-matter Interactions and Quantum Information Processing (United States)

    Gao, Jie

    Chip-scale photonic devices such as microdisks, photonic crystal cavities and slow-light photonic crystal waveguides possess strong light localization and long photon lifetime, which will significantly enhance the light-matter interactions and can be used to implement new functionalities for both classical and quantum information processing, optical computation and optical communication in integrated nanophotonic circuits. This thesis will focus on three topics about light matter interactions and quantum information processing with chip-scale photonic devices, including 1) Design and characterization of asymmetric resonate cavity with radiation directionality and air-slot photonic crystal cavity with ultrasmall effective mode volume, 2) Exciton-photon interactions between quantum dots and photonic crystal devices and non-classical photon source from a single quantum dot, and 3) Quantum controlled phase gate and phase switching based on quantum dots and photonic crystal waveguide. The first topic is engineered control of radiation directionality and effective mode volume for optical mode in chip-scale silicon micro-/nano-cavities. High quality factor (Q), subwavelength mode volume ( V) and controllable radiation directionality are the major properties for optical cavities designs. In Chapter 2, asymmetric resonant cavities with rational caustics are proposed and interior whispering gallery modes in monolithic silicon mesoscopic microcavities are experimentally demonstrated. These microcavities possess unique robustness of cavity quality factor against roughness Rayleigh scattering. In Chapter 3, air-slot mode-gap photonic crystal cavities with quality factor of 104 and effective mode volume ˜ 0.02 cubic wavelengths are experimentally demonstrated. The origin of the high Q air-slot cavity mode is the mode-gap effect from the slotted photonic crystal waveguide mode with negative dispersion. The second topic is exciton-photon coupling between quantum dots and

  18. Evaluation and QSAR modeling on multiple endpoints of estrogen activity based on different bioassays. (United States)

    Liu, Huanxiang; Papa, Ester; Gramatica, Paola


    There is a great need for an effective means of rapidly assessing endocrine-disrupting activity, especially estrogen-simulating activity, due to the large number of chemicals that have serious adverse effects on the environment. Many approaches using a variety of biological screening assays are used to identify endocrine disrupting chemicals. The present investigation analyzes the consistency and peculiarity of information from different experimental assays collected from a literature survey, by studying the correlation of the different endpoints. In addition, the activity values of more widely used selected bioassays have been combined by principle components analysis (PCA) to build one cumulative endpoint, the estrogen activity index (EAI), for priority setting to identify chemicals most likely possessing estrogen activity for early entry into screening. This index was then modeled using only a few theoretical molecular descriptors. The constructed MLR-QSAR model has been statistically validated for its predictive power, and can be proposed as a preliminary evaluative method to screen/prioritize estrogens according to their integrated estrogen activity, just starting from molecular structure.

  19. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

    Directory of Open Access Journals (Sweden)

    Emily Peak

    Full Text Available BACKGROUND: Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. METHODOLOGY/PRINCIPAL FINDINGS: We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase, praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed, relevant to industrial (384-well microtiter plate compatibility and academic (96-well microtiter plate compatibility settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative. CONCLUSIONS/SIGNIFICANCE: The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat

  20. Label-Free and Separation-Free Atomic Fluorescence Spectrometry-Based Bioassay: Sensitive Determination of Single-Strand DNA, Protein, and Double-Strand DNA. (United States)

    Chen, Piaopiao; Wu, Peng; Chen, Junbo; Yang, Peng; Zhang, Xinfeng; Zheng, Chengbin; Hou, Xiandeng


    Based on selective and sensitive determination of Hg(2+) released from mercury complex by cold vapor generation (CVG) atomic fluorescence spectrometry (AFS) using SnCl2 as a reductant, a novel label-free and separation-free strategy was proposed for DNA and protein bioassay. To construct the DNA bioassay platform, an Hg(2+)-mediated molecular beacon (hairpin) without labeling but possessing several thymine (T) bases at both ends was employed as the probe. It is well-known that Hg(2+) could trigger the formation of the hairpin structure through T-Hg(2+)-T connection. In the presence of a specific target, the hairpin structure could be broken and the captured Hg(2+) was released. Interestingly, it was found that SnCl2 could selectively reduce only free Hg(2+) to Hg(0) vapor in the presence of T-Hg(2+)-T complex, which could be separated from sample matrices for sensitive AFS detection. Three different types of analyte, namely, single-strand DNA (ssDNA), protein, and double-strand DNA (dsDNA), were investigated as the target analytes. Under the optimized conditions, this bioassay provided high sensitivity for ssDNA, protein, and dsDNA determination with the limits of detection as low as 0.2, 0.08, and 0.3 nM and the linear dynamic ranges of 10-150, 5-175, and 1-250 nM, respectively. The analytical performance for these analytes compares favorably with those by previously reported methods, demonstrating the potential usefulness and versatility of this new AFS-based bioassay. Moreover, the bioassay retains advantages of simplicity, cost-effectiveness, and sensitivity compared to most of the conventional methods.

  1. A choline oxidase amperometric bioassay for the detection of mustard agents based on screen-printed electrodes modified with Prussian Blue nanoparticles. (United States)

    Arduini, Fabiana; Scognamiglio, Viviana; Covaia, Corrado; Amine, Aziz; Moscone, Danila; Palleschi, Giuseppe


    In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (-50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.

  2. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    DEFF Research Database (Denmark)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen


    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  3. Rapid, label-free, electrical whole blood bioassay based on nanobiosensor systems. (United States)

    Chang, Hsiao-Kang; Ishikawa, Fumiaki N; Zhang, Rui; Datar, Ram; Cote, Richard J; Thompson, Mark E; Zhou, Chongwu


    Biomarker detection based on nanowire biosensors has attracted a significant amount of research effort in recent years. However, only very limited research work has been directed toward biomarker detection directly from physiological fluids mainly because of challenges caused by the complexity of media. This limitation significantly reduces the practical impact generated by the aforementioned nanobiosensors. In this study, we demonstrate an In(2)O(3) nanowire-based biosensing system that is capable of performing rapid, label-free, electrical detection of cancer biomarkers directly from human whole blood collected by a finger prick. Passivating the nanowire surface successfully blocked the signal induced by nonspecific binding when performing active measurement in whole blood. Passivated devices showed markedly smaller signals induced by nonspecific binding of proteins and other biomaterials in serum and higher sensitivity to target biomarkers than bare devices. The detection limit of passivated sensors for biomarkers in whole blood was similar to the detection limit for the same analyte in purified buffer solutions at the same ionic strength, suggesting minimal decrease in device performance in the complex media. We then demonstrated detection of multiple cancer biomarkers with high reliability at clinically meaningful concentrations from whole blood collected by a finger prick using this sensing system.

  4. Application of the yeast-based reporter gene bioassay for the assessment of estrogenic activity in cow's milk from Poland. (United States)

    Stypuła-Trębas, Sylwia; Minta, Maria; Radko, Lidia; Żmudzki, Jan


    Milk contain compounds acting through the estrogen receptor signaling. The still open question whether such estrogens pose a risk for human health, encouraged us to measure the overall estrogenic activity of cow's milk in the in vitro yeast reporter bioassay. First, we assessed the ability of the bioassay to detect estrogens frequently detected in milk. The relative potencies of 16 compounds descended in the order: 17β-estradiol (17β-E2), 17α-ethinylestradiol, diethylstilbestrol, dienestrol, 17α-E2, estrone, zearalenone, estriol, equol, genistein, 17β-E2 glucuronide, bisphenol A, apigenin, daidzein. Flavone, 4-n-nonylphenol and 4-t-octylphenol shown no activity in the bioassay.The estrogenic activities of milk samples without hydrolysis were below the detection limit, whereas in 50% of the deconjugated samples they varied between 0.29 and 0.49 ng EEQ mL(-1). We also compared the estrogenic activity in raw cow's milk collected from rural and industrial locations in Poland. In our pilot study we did not observe statistically significant difference in estrogenic activities in milk collected from the two locations. We found that the daily intake of estrogens with milk may be higher than estrogen levels in human serum. Further studies are warranted to evaluate the significance of milk and dairy as a source of estrogens for humans.

  5. Chip-scale hermetic feedthroughs for implantable bionics. (United States)

    Guenther, Thomas; Dodds, Christopher W D; Lovell, Nigel H; Suaning, Gregg J


    Most implantable medical devices such as cochlear implants and visual prostheses require protection of the stimulating electronics. This is achieved by way of a hermetic feedthrough system which typically features three important attributes: biocompatibility with the human body, device hermeticity and density of feedthrough conductors. On the quest for building a visual neuroprosthesis, a high number of stimulating channels is required. This has encouraged new technologies with higher rates of production yield and further miniaturization. An Al(2)O(3) based feedthrough system has been developed comprising up to 20 platinum feedthroughs per square millimeter. Ceramics substrates are shown to have leak rates below 1 × 10(-12) atm × cc/s, thus exceeding the resolution limits of most commercially available leak detectors. A sheet resistance of 0.05 Ω can be achieved. This paper describes the design, fabrication process and hermeticity testing of high density feedthroughs for use in neuroprosthetic implants.

  6. SPIDER: Next Generation Chip Scale Imaging Sensor Update (United States)

    Duncan, A.; Kendrick, R.; Ogden, C.; Wuchenich, D.; Thurman, S.; Su, T.; Lai, W.; Chun, J.; Li, S.; Liu, G.; Yoo, S. J. B.


    The Lockheed Martin Advanced Technology Center (LM ATC) and the University of California at Davis (UC Davis) are developing an electro-optical (EO) imaging sensor called SPIDER (Segmented Planar Imaging Detector for Electro-optical Reconnaissance) that seeks to provide a 10x to 100x size, weight, and power (SWaP) reduction alternative to the traditional bulky optical telescope and focal-plane detector array. The substantial reductions in SWaP would reduce cost and/or provide higher resolution by enabling a larger-aperture imager in a constrained volume. Our SPIDER imager replaces the traditional optical telescope and digital focal plane detector array with a densely packed interferometer array based on emerging photonic integrated circuit (PIC) technologies that samples the object being imaged in the Fourier domain (i.e., spatial frequency domain), and then reconstructs an image. Our approach replaces the large optics and structures required by a conventional telescope with PICs that are accommodated by standard lithographic fabrication techniques (e.g., complementary metal-oxide-semiconductor (CMOS) fabrication). The standard EO payload integration and test process that involves precision alignment and test of optical components to form a diffraction limited telescope is, therefore, replaced by in-process integration and test as part of the PIC fabrication, which substantially reduces associated schedule and cost. This paper provides an overview of performance data on the second-generation PIC for SPIDER developed under the Defense Advanced Research Projects Agency (DARPA)'s SPIDER Zoom research funding. We also update the design description of the SPIDER Zoom imaging sensor and the second-generation PIC (high- and low resolution versions).

  7. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    Energy Technology Data Exchange (ETDEWEB)

    Ni, Jing [Iowa State Univ., Ames, IA (United States)


    This work describes several research projects aimed towards developing new instruments and novel methods for high throughput chemical and biological analysis. Approaches are taken in two directions. The first direction takes advantage of well-established semiconductor fabrication techniques and applies them to miniaturize instruments that are workhorses in analytical laboratories. Specifically, the first part of this work focused on the development of micropumps and microvalves for controlled fluid delivery. The mechanism of these micropumps and microvalves relies on the electrochemically-induced surface tension change at a mercury/electrolyte interface. A miniaturized flow injection analysis device was integrated and flow injection analyses were demonstrated. In the second part of this work, microfluidic chips were also designed, fabricated, and tested. Separations of two fluorescent dyes were demonstrated in microfabricated channels, based on an open-tubular liquid chromatography (OT LC) or an electrochemically-modulated liquid chromatography (EMLC) format. A reduction in instrument size can potentially increase analysis speed, and allow exceedingly small amounts of sample to be analyzed under diverse separation conditions. The second direction explores the surface enhanced Raman spectroscopy (SERS) as a signal transduction method for immunoassay analysis. It takes advantage of the improved detection sensitivity as a result of surface enhancement on colloidal gold, the narrow width of Raman band, and the stability of Raman scattering signals to distinguish several different species simultaneously without exploiting spatially-separated addresses on a biochip. By labeling gold nanoparticles with different Raman reporters in conjunction with different detection antibodies, a simultaneous detection of a dual-analyte immunoassay was demonstrated. Using this scheme for quantitative analysis was also studied and preliminary dose-response curves from an immunoassay of a

  8. A chip-scale, telecommunications-band frequency conversion interface for quantum emitters. (United States)

    Agha, Imad; Ates, Serkan; Davanço, Marcelo; Srinivasan, Kartik


    We describe a chip-scale, telecommunications-band frequency conversion interface designed for low-noise operation at wavelengths desirable for common single photon emitters. Four-wave-mixing Bragg scattering in silicon nitride waveguides is used to demonstrate frequency upconversion and downconversion between the 980 nm and 1550 nm wavelength regions, with signal-to-background levels > 10 and conversion efficiency of ≈ -60 dB at low continuous wave input pump powers ( 25 % in existing geometries. Finally, we present waveguide designs that can be used to connect shorter wavelength (637 nm to 852 nm) quantum emitters with 1550 nm.

  9. Light-Addressed Electrodeposition of Enzyme-Entrapped Chitosan Membranes for Multiplexed Enzyme-Based Bioassays Using a Digital Micromirror Device

    Directory of Open Access Journals (Sweden)

    Yeu-Long Jiang


    Full Text Available This paper describes a light-addressed electrolytic system used to perform an electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassays using a digital micromirror device (DMD. In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-cathode to electrolytically produce hydroxide ions, which leads to an increased pH gradient. The high pH generated at the cathode can cause a local gelation of chitosan through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressed electrodeposition of chitosan membranes with different shapes and sizes, as well as multiplexed micropatterning, was performed. The effect of the illumination time of the light pattern on the dimensional resolution of chitosan membrane formation was examined experimentally. Moreover, multiplexed enzyme-based bioassay of enzyme-entrapped chitosan membranes was also successfully demonstrated through the electrodeposition of the chitosan membranes with various shapes/sizes and entrapping different enzymes. As a model experiment, glucose and ethanol were simultaneously detected in a single detection chamber without cross-talk using shape-coded chitosan membranes entrapped with glucose oxidase (GOX, peroxidase (POD, and Amplex Red (AmR or alcohol oxidase (AOX, POD, and AmR by using same fluorescence indicator (AmR.

  10. Investigation on pattern and methods of quality control for Chinese materia medica based on Dao-di herbs and bioassay-bioassay for Coptis chinensis%基于道地药材和生物测定的中药质量控制模式与方法研究——黄连质量生物测定

    Institute of Scientific and Technical Information of China (English)

    鄢丹; 肖小河


    针对中药质量生物评价与控制中面临的重要技术问题--生物测定方法,以黄连质量生物评价与控制(抑菌活性)为代表性实例,从方法学内涵、选择原则、实验设计、数据分析等方面,对融合生物效价值和生物效应谱表征技术为一体的抑菌活性检测的新方法建立过程及应用进行阐释,以期为构建基于道地药材和生物测定的中药质量控制模式与方法提供技术参考.%Estaolishment of bioassay methods is the technical issues to be faced with in the bioassay of Chinese materia medica. Taking the bioassay of Coptis chinensis Franch as an example, the establishment process and application of the bioassay methods (including bio-potency and bio-activity fingerprint) were explained from the aspects of methodology, principle of selection, experimental design, method confirmation and data analysis. The common technologies were extracted and formed with the above aspects, so as to provide technical support for constructing pattern and method of the quality control for Chinese materia medica based on the Dao-di herbs and bioassay.

  11. A new P. putida instrumental toxicity bioassay. (United States)

    Figueredo, Federico; Abrevaya, Ximena C; Cortón, Eduardo


    Here, we present a new toxicity bioassay (CO2-TOX), able to detect toxic or inhibitory compounds in water samples, based on the quantification of Pseudomonas putida KT2440 CO2 production. The metabolically produced CO2 was measured continuously and directly in the liquid assay media, with a potentiometric gas electrode. The optimization studies were performed using as a model toxicant 3,5-DCP (3,5-dichlorophenol); later, heavy metals (Pb(2+), Cu(2+), or Zn(2+)) and a metalloid (As(5+)) were assayed. The response to toxics was evident after 15 min of incubation and at relatively low concentrations (e.g., 1.1 mg/L of 3,5-DCP), showing that the CO2-TOX bioassay is fast and sensitive. The EC50 values obtained were 4.93, 0.12, 6.05, 32.17, and 37.81 mg/L for 3,5-DCP, Cu(2+), Zn(2+), As(5+), and Pb(2+), respectively, at neutral pH. Additionally, the effect of the pH of the sample and the use of lyophilized bacteria were also analyzed showing that the bioassay can be implemented in different conditions. Moreover, highly turbid samples and samples with very low oxygen levels were measured successfully with the new instrumental bioassay described here. Finally, simulated samples containing 3,5-DCP or a heavy metal mixture were tested using the proposed bioassay and a standard ISO bioassay, showing that our test is more sensible to the phenol but less sensible to the metal mixtures. Therefore, we propose CO2-TOX as a rapid, sensitive, low-cost, and robust instrumental bioassay that could perform as an industrial wastewater-process monitor among other applications.

  12. Water Powered Bioassay System (United States)


    capillary micropump 27 Figure 30: Slow dripping/separation of a droplet from a capillary 4.1.5 Micro Osmotic Pumping Nano Droplet...stored and delivered fluidic pressure and, with a combination of pumps and valves, formed the basic micro fluidic processing unit. The addition of...System, Microvalve, Micro -Accumulator, Micro Dialysis Needle, Bioassay System, Water Activated, Micro Osmotic Pump 16. PRICE CODE 17. SECURITY

  13. A scalable silicon photonic chip-scale optical switch for high performance computing systems. (United States)

    Yu, Runxiang; Cheung, Stanley; Li, Yuliang; Okamoto, Katsunari; Proietti, Roberto; Yin, Yawei; Yoo, S J B


    This paper discusses the architecture and provides performance studies of a silicon photonic chip-scale optical switch for scalable interconnect network in high performance computing systems. The proposed switch exploits optical wavelength parallelism and wavelength routing characteristics of an Arrayed Waveguide Grating Router (AWGR) to allow contention resolution in the wavelength domain. Simulation results from a cycle-accurate network simulator indicate that, even with only two transmitter/receiver pairs per node, the switch exhibits lower end-to-end latency and higher throughput at high (>90%) input loads compared with electronic switches. On the device integration level, we propose to integrate all the components (ring modulators, photodetectors and AWGR) on a CMOS-compatible silicon photonic platform to ensure a compact, energy efficient and cost-effective device. We successfully demonstrate proof-of-concept routing functions on an 8 × 8 prototype fabricated using foundry services provided by OpSIS-IME.

  14. A stabilized chip-scale Kerr frequency comb via a high-Q reference photonic microresonator

    CERN Document Server

    Lim, Jinkang; Vinod, Abhinav K; Mortazavian, Parastou; Yu, Mingbin; Kwong, Dim-Lee; Savchenkov, Anatoliy A; Matsko, Andrey B; Maleki, Lute; Wong, Chee Wei


    We stabilize a chip-scale Si3N4 phase-locked Kerr frequency comb via locking the pump laser to an independent stable high-Q reference microresonator and locking the comb spacing to an external microwave oscillator. In this comb, the pump laser shift induces negligible impact on the comb spacing change. This scheme is a step towards miniaturization of the stabilized Kerr comb system as the microresonator reference can potentially be integrated on-chip. Fractional instability of the optical harmonics of the stabilized comb is limited by the microwave oscillator used for comb spacing lock below 1 s averaging time and coincides with the pump laser drift in the long term.

  15. A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon.

    Directory of Open Access Journals (Sweden)

    Marianne Berger Rentsch

    Full Text Available Type I interferons (IFN comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29, a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.


    Energy Technology Data Exchange (ETDEWEB)

    Vormelker, P


    Two high-pressure bioassay vessels failed at the Savannah River Site during a microwave heating process for biosample testing. Improper installation of the thermal shield in the first failure caused the vessel to burst during microwave heating. The second vessel failure is attributed to overpressurization during a test run. Vessel failure appeared to initiate in the mold parting line, the thinnest cross-section of the octagonal vessel. No material flaws were found in the vessel that would impair its structural performance. Content weight should be minimized to reduce operating temperature and pressure. Outer vessel life is dependent on actual temperature exposure. Since thermal aging of the vessels can be detrimental to their performance, it was recommended that the vessels be used for a limited number of cycles to be determined by additional testing.

  17. Estrogen Receptor Agonists and Antagonists in the Yeast Estrogen Bioassay. (United States)

    Wang, Si; Bovee, Toine F H


    Cell-based bioassays can be used to predict the eventual biological activity of a substance on a living organism. In vitro reporter gene bioassays are based on recombinant vertebrate cell lines or yeast strains and especially the latter are easy-to-handle, cheap, and fast. Moreover, yeast cells do not express estrogen, androgen, progesterone or glucocorticoid receptors, and are thus powerful tools in the development of specific reporter gene systems that are devoid of crosstalk from other hormone pathways. This chapter describes our experience with an in-house developed RIKILT yeast estrogen bioassay for testing estrogen receptor agonists and antagonists, focusing on the applicability of the latter.

  18. Reliability and Characteristics of Wafer-Level Chip-Scale Packages under Current Stress (United States)

    Chen, Po-Ying; Kung, Heng-Yu; Lai, Yi-Shao; Hsiung Tsai, Ming; Yeh, Wen-Kuan


    In this work, we present a novel approach and method for elucidating the characteristics of wafer-level chip-scale packages (WLCSPs) for electromigration (EM) tests. The die in WLCSP was directly attached to the substrate via a soldered interconnect. The shrinking of the area of the die that is available for power, and the solder bump also shrinks the volume and increases the density of electrons for interconnect efficiency. The bump current density now approaches to 106 A/cm2, at which point the EM becomes a significant reliability issue. As known, the EM failure depends on numerous factors, including the working temperature and the under bump metallization (UBM) thickness. A new interconnection geometry is adopted extensively with moderate success in overcoming larger mismatches between the displacements of components during current and temperature changes. Both environments and testing parameters for WLCSP are increasingly demanded. Although failure mechanisms are considered to have been eliminated or at least made manageable, new package technologies are again challenging its process, integrity and reliability. WLCSP technology was developed to eliminate the need for encapsulation to ensure compatibility with smart-mount technology (SMT). The package has good handing properties but is now facing serious reliability problems. In this work, we investigated the reliability of a WLCSP subjected to different accelerated current stressing conditions at a fixed ambient temperature of 125 °C. A very strong correlation exists between the mean time to failure (MTTF) of the WLCSP test vehicle and the mean current density that is carried by a solder joint. A series of current densities were applied to the WLCSP architecture; Black's power law was employed in a failure mode simulation. Additionally, scanning electron microscopy (SEM) was adopted to determine the differences existing between high- and low-current-density failure modes.

  19. Surface-Enhanced Raman Spectroscopy Based Quantitative Bioassay on Aptamer-Functionalized Nanopillars Using Large-Area Raman Mapping

    DEFF Research Database (Denmark)

    Yang, Jaeyoung; Palla, Mirko; Bosco, Filippo;


    Surface-enhanced Raman spectroscopy (SERS) has been used in a variety of biological applications due to its high sensitivity and specificity. Here, we report a SERS-based biosensing approach for quantitative detection of biomolecules. A SERS substrate bearing gold-decorated silicon nanopillars is...

  20. Rapid screening of aquatic toxicity of several metal-based nanoparticles using the MetPLATE Trade-Mark-Sign bioassay

    Energy Technology Data Exchange (ETDEWEB)

    Pokhrel, Lok R.; Silva, Thilini [Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN 37614 (United States); Dubey, Brajesh, E-mail: [Environmental Engineering, School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario (Canada); El Badawy, Amro M. [Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, OH (United States); Tolaymat, Thabet M. [USEPA, Office of Research and Development, National Risk Management Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45224 (United States); Scheuerman, Phillip R. [Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN 37614 (United States)


    Current understanding of potential toxicity of engineered nanomaterials to aquatic microorganisms is limited for risk assessment and management. Here we evaluate if the MetPLATE Trade-Mark-Sign test can be used as an effective and rapid screening tool to test for potential aquatic toxicity of various metal-based nanoparticles (NPs). The MetPLATE bioassay is a heavy metal sensitive test based on {beta}-galactosidase activity in Escherichia coli. Five different types of metal-based NPs were screened for toxicity: (1) citrate coated nAg (Citrate-nanosilver), (2) polyvinylpyrrolidone coated nAg (PVP-nAg), (3) uncoated nZnO, (4) uncoated nTiO{sub 2} and (5) 1-Octadecylamine coated CdSe Quantum Dots (CdSe QDs); and compared with their corresponding ionic salt toxicity. Citrate-nAg was further fractionated into clean Citrate-nAg, unclean Citrate-nAg and permeate using a tangential flow filtration (TFF) system to eliminate residual ions and impurities from the stock Citrate-nAg suspension and also to differentiate between ionic- versus nano-specific toxicity. Our results showed that nAg, nZnO and CdSe QDs were less toxic than their corresponding ionic salts tested, while nano- or ionic form of TiO{sub 2} was not toxic as high as 2.5 g L{sup -1} to the MetPLATE Trade-Mark-Sign bacteria. Although coating-dependent toxicity was noticeable between two types of Ag NPs evaluated, particle size and surface charge were not adequate to explain the observed toxicity; hence, the toxicity appeared to be material-specific. Overall, the toxicity followed the trend: CdCl{sub 2} > AgNO{sub 3} > PVP-nAg > unclean Citrate-nAg > clean Citrate-nAg > ZnSO{sub 4} > nZnO > CdSe QDs > nTiO{sub 2}/TiO{sub 2}. These results indicate that an evaluation of {beta}-galactosidase inhibition in MetPLATE Trade-Mark-Sign E. coli can be an important consideration for rapid screening of metal-based NP toxicity, and should facilitate ecological risk assessment of these emerging contaminants. - Highlights

  1. A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions

    Directory of Open Access Journals (Sweden)

    Robert Kraft


    The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the ‘filagree’ phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity. The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the ‘beads-on-a-string’ defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

  2. Giant Magnetoresistive Sensors and Magnetic Labels for Chip-Scale Detection of Immunosorbent Assays

    Energy Technology Data Exchange (ETDEWEB)

    Rachel Lora Millen


    The combination of giant magnetoresistive sensors, magnetic labeling strategies, and biomolecule detection is just beginning to be explored. New readout methods and assay formats are necessary for biomolecules detection to flourish. The work presented in this dissertation describes steps toward the creation of a novel detection method for bioassays utilizing giant magnetoresistive sensors as the readout method. The introduction section contains a brief review of some of the current methods of bioassay readout. The theoretical underpinnings of the giant magnetoresistive effect are also discussed. Finally, the more prominent types of giant magnetoresistive sensors are described, as well as their complicated fabrication. Four data chapters follow the introduction; each chapter is presented as a separate manuscript, either already published or soon to be submitted. Chapter 1 presents research efforts toward the production of a bioassay on the surface of a gold-modified GMR sensor. The testing of this methodology involved the capture of goat a-mouse-coated magnetic nanoparticles on the mouse IgG-modified gold surface. The second, third and fourth chapters describe the utilization of a self-referenced sample stick for scanning across the GMR sensor. The sample stick consisted of alternating magnetic reference and bioactive gold addresses. Chapter 2 is concerned with the characterization of both the scanning readout method and the binding and detection of streptavidin-coated magnetic particles to a biotinylated surface. Chapter 3 advances the sample stick readout with the use of the system for detection of a sandwich immunoassay with rabbit IgG proteins. Finally, simultaneous detection of three IgG proteins is demonstrated in Chapter 4. The dissertation is concluded with a brief summary of the research presented and a discussion of the possible future applications and direction of this work.

  3. Giant Magnetoresistive Sensors and Magnetic Labels for Chip-Scale Detection of Immunosorbent Assays

    Energy Technology Data Exchange (ETDEWEB)

    Millen, Rachel Lora [Iowa State Univ., Ames, IA (United States)


    The combination of giant magnetoresistive sensors, magnetic labeling strategies, and biomolecule detection is just beginning to be explored. New readout methods and assay formats are necessary for biomolecules detection to flourish. The work presented in this dissertation describes steps toward the creation of a novel detection method for bioassays utilizing giant magnetoresistive sensors as the readout method. The introduction section contains a brief review of some of the current methods of bioassay readout. The theoretical underpinnings of the giant magnetoresistive effect are also discussed. Finally, the more prominent types of giant magnetoresistive sensors are described, as well as their complicated fabrication. Four data chapters follow the introduction; each chapter is presented as a separate manuscript, either already published or soon to be submitted. Chapter 1 presents research efforts toward the production of a bioassay on the surface of a gold-modified GMR sensor. The testing of this methodology involved the capture of goat a-mouse-coated magnetic nanoparticles on the mouse IgG-modified gold surface. The second, third and fourth chapters describe the utilization of a self-referenced sample stick for scanning across the GMR sensor. The sample stick consisted of alternating magnetic reference and bioactive gold addresses. Chapter 2 is concerned with the characterization of both the scanning readout method and the binding and detection of streptavidin-coated magnetic particles to a biotinylated surface. Chapter 3 advances the sample stick readout with the use of the system for detection of a sandwich immunoassay with rabbit IgG proteins. Finally, simultaneous detection of three IgG proteins is demonstrated in Chapter 4. The dissertation is concluded with a brief summary of the research presented and a discussion of the possible future applications and direction of this work.

  4. Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine

    NARCIS (Netherlands)

    Bovee, T.F.H.; Heskamp, H.H.; Hamers, A.R.M.; Hoogenboom, L.A.P.; Nielen, M.W.F.


    Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor a (hERa) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, this yeast estrogen assay was validated as a qualitative screening

  5. Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay. (United States)

    Reverté, Laia; de la Iglesia, Pablo; del Río, Vanessa; Campbell, Katrina; Elliott, Christopher T; Kawatsu, Kentaro; Katikou, Panagiota; Diogène, Jorge; Campàs, Mònica


    The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

  6. Bioassay Phantoms Using Medical Images and Computer Aided Manufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Dr. X. Geroge Xu


    A radiation bioassay program relies on a set of standard human phantoms to calibrate and assess radioactivity levels inside a human body for radiation protection and nuclear medicine imaging purposes. However, the methodologies in the development and application of anthropomorphic phantoms, both physical and computational, had mostly remained the same for the past 40 years. We herein propose a 3-year research project to develop medical image-based physical and computational phantoms specifically for radiation bioassay applications involving internally deposited radionuclides. The broad, long-term objective of this research was to set the foundation for a systematic paradigm shift away from the anatomically crude phantoms in existence today to realistic and ultimately individual-specific bioassay methodologies. This long-term objective is expected to impact all areas of radiation bioassay involving nuclear power plants, U.S. DOE laboratories, and nuclear medicine clinics.

  7. Microwave-accelerated bioassay technique for rapid and quantitative detection of biological and environmental samples. (United States)

    Mohammed, Muzaffer; Syed, Maleeha F; Aslan, Kadir


    Quantitative detection of molecules of interest from biological and environmental samples in a rapid manner, particularly with a relevant concentration range, is imperative to the timely assessment of human diseases and environmental issues. In this work, we employed the microwave-accelerated bioassay (MAB) technique, which is based on the combined use of circular bioassay platforms and microwave heating, for rapid and quantitative detection of Glial Fibrillary Acidic Protein (GFAP) and Shiga like toxin (STX 1). The proof-of-principle use of the MAB technique with the circular bioassay platforms for the rapid detection of GFAP in buffer based on colorimetric and fluorescence readouts was demonstrated with a 900W kitchen microwave. We also employed the MAB technique with a new microwave system (called the iCrystal system) for the detection of GFAP from mice with brain injuries and STX 1 from a city water stream. Control bioassays included the commercially available gold standard bioassay kits run at room temperature. Our results show that the lower limit of detection (LLOD) of the colorimetric and fluorescence based bioassays for GFAP was decreased by ~1000 times using the MAB technique and our circular bioassay platforms as compared to the commercially available bioassay kits. The overall bioassay time for GFAP and STX 1 was reduced from 4h using commercially available bioassay kits to 10min using the MAB technique.

  8. Evaluation of effectiveness of entomopathogenic fungi Beauveria bassiana using a standard laboratory bioassay



    In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the yellow mealworm (Tenebrio molitor) was tested under various temperature conditions. Six different strains of fungus B. bassiana was investigated. The evaluation was based on vitality bioassays including germination and growth index assessment and the bioassay of virulence based on target organism T. molitor was also assessed growth and yield of conidia different strains of fungus B. bassiana on...

  9. A broadband chip-scale optical frequency synthesizer at 2.7 × 10(-16) relative uncertainty. (United States)

    Huang, Shu-Wei; Yang, Jinghui; Yu, Mingbin; McGuyer, Bart H; Kwong, Dim-Lee; Zelevinsky, Tanya; Wong, Chee Wei


    Optical frequency combs-coherent light sources that connect optical frequencies with microwave oscillations-have become the enabling tool for precision spectroscopy, optical clockwork, and attosecond physics over the past decades. Current benchmark systems are self-referenced femtosecond mode-locked lasers, but Kerr nonlinear dynamics in high-Q solid-state microresonators has recently demonstrated promising features as alternative platforms. The advance not only fosters studies of chip-scale frequency metrology but also extends the realm of optical frequency combs. We report the full stabilization of chip-scale optical frequency combs. The microcomb's two degrees of freedom, one of the comb lines and the native 18-GHz comb spacing, are simultaneously phase-locked to known optical and microwave references. Active comb spacing stabilization improves long-term stability by six orders of magnitude, reaching a record instrument-limited residual instability of [Formula: see text]. Comparing 46 nitride frequency comb lines with a fiber laser frequency comb, we demonstrate the unprecedented microcomb tooth-to-tooth relative frequency uncertainty down to 50 mHz and 2.7 × 10(-16), heralding novel solid-state applications in precision spectroscopy, coherent communications, and astronomical spectrography.

  10. A broadband chip-scale optical frequency synthesizer at 2.7 × 10−16 relative uncertainty (United States)

    Huang, Shu-Wei; Yang, Jinghui; Yu, Mingbin; McGuyer, Bart H.; Kwong, Dim-Lee; Zelevinsky, Tanya; Wong, Chee Wei


    Optical frequency combs—coherent light sources that connect optical frequencies with microwave oscillations—have become the enabling tool for precision spectroscopy, optical clockwork, and attosecond physics over the past decades. Current benchmark systems are self-referenced femtosecond mode-locked lasers, but Kerr nonlinear dynamics in high-Q solid-state microresonators has recently demonstrated promising features as alternative platforms. The advance not only fosters studies of chip-scale frequency metrology but also extends the realm of optical frequency combs. We report the full stabilization of chip-scale optical frequency combs. The microcomb’s two degrees of freedom, one of the comb lines and the native 18-GHz comb spacing, are simultaneously phase-locked to known optical and microwave references. Active comb spacing stabilization improves long-term stability by six orders of magnitude, reaching a record instrument-limited residual instability of 3.6mHz/τ. Comparing 46 nitride frequency comb lines with a fiber laser frequency comb, we demonstrate the unprecedented microcomb tooth-to-tooth relative frequency uncertainty down to 50 mHz and 2.7 × 10−16, heralding novel solid-state applications in precision spectroscopy, coherent communications, and astronomical spectrography. PMID:27152341

  11. Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum. (United States)

    Chen, Xinyi C; Zhou, Lei; Gupta, Shalini; Civoli, Francesca


    The administration of biological therapeutics can potentially elicit the development of neutralizing antibodies (NAbs) to the therapeutic drug in patients, which could have a significant impact on drug efficacy and safety. A rigorous in vitro cell-based assay for the detection of NAbs is critical for the assessment of the immunogenicity profile of the therapeutic drug. Conatumumab is a fully human monoclonal agonist antibody directed against the extracellular domain of human TRAIL receptor 2 (TR-2). It is being investigated as a cancer treatment because it is able to induce apoptosis in sensitive tumor cells. This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experiments for assay optimization and 1 DOE experiment to validate assay robustness. We evaluated key operating parameters of the assay such as cell number, percentage of serum matrix, concentration of the therapeutic drug, concentration of the cross-linker, length of various incubation steps, cell age, interval between cell subculture and bioassay time, and detection equipment.

  12. 77 FR 14837 - Bioassay at Uranium Mills (United States)


    ... COMMISSION Bioassay at Uranium Mills AGENCY: Nuclear Regulatory Commission. ACTION: Draft regulatory guide... for public comment draft regulatory guide (DG), DG-8051, ``Bioassay at Uranium Mills.'' This guide describes a bioassay program acceptable to the NRC staff for uranium mills and applicable portions...

  13. A rapid resazurin bioassay for assessing the toxicity of fungicides. (United States)

    Fai, Patricia Bi; Grant, Alastair


    Fungicides are widely used in agriculture, and released in large amounts to the environment. Methods used for antifungal susceptibility testing are cumbersome and time-consuming. As a result, very little attention has been paid to including fungal tests in the routine screening of pesticides and there are no reports in the literature of fungicide focussed effects directed analysis (EDA). In addition very little is known on the toxicity of fungicides to environmentally significant fungi. Here we report a rapid microplate-based resorufin fluorescence inhibition bioassay and compare it with a 24h microplate-based yeast growth inhibition bioassay using eight fungicides. The growth inhibition bioassay was sensitive, giving IC50 and IC90 values comparable to previously reported IC50 or MICs of these fungicides for Saccharomyces cerevisiae and other fungi. The resorufin fluorescence inhibition bioassay was both faster and more sensitive than the growth inhibition bioassay. Inhibitory concentrations obtained just after 30min of incubation with amphotericin B (AMB) and captan were at least a hundred fold lower than IC50s in the literature for fungi. The fluorescence bioassay showed only a small response to pyrazophos and thiabendazole but these only inhibited growth at high concentrations so this may reflect low sensitivity of S. cerevisiae to these particular fungicides. This bioassay can detect toxic effects of a range of fungicides from different chemical classes with different modes of action. It will be valuable for screening chemical libraries for fungicides and as a biomarker for detecting the effects of fungicides to non-target fungi.

  14. Individual Radiological Protection Monitoring of Utrok Atoll Residents Based on Whole Body Counting of Cesium-137 (137Cs) and Plutonium Bioassay

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, T; Kehl, S; Brown, T; Martinelli, R; Hickman, D; Jue, T; Tumey, S; Langston, R


    This report contains individual radiological protection surveillance data developed during 2006 for adult members of a select group of families living on Utrok Atoll. These Group I volunteers all underwent a whole-body count to determine levels of internally deposited cesium-137 ({sup 137}Cs) and supplied a bioassay sample for analysis of plutonium isotopes. Measurement data were obtained and the results compared with an equivalent set of measurement data for {sup 137}Cs and plutonium isotopes from a second group of adult volunteers (Group II) who were long-term residents of Utrok Atoll. For the purposes of this comparison, Group II volunteers were considered representative of the general population on Utrok Atoll. The general aim of the study was to determine residual systemic burdens of fallout radionuclides in each volunteer group, develop data in response to addressing some specific concerns about the preferential uptake and potential health consequences of residual fallout radionuclides in Group I volunteers, and generally provide some perspective on the significance of radiation doses delivered to volunteers (and the general Utrok Atoll resident population) in terms of radiological protection standards and health risks. Based on dose estimates from measurements of internally deposited {sup 137}Cs and plutonium isotopes, the data and information developed in this report clearly show that neither volunteer group has acquired levels of internally deposited fallout radionuclides specific to nuclear weapons testing in the Marshall Islands that are likely to have any consequence on human health. Moreover, the dose estimates are well below radiological protection standards as prescribed by U.S. regulators and international agencies, and are very small when compared to doses from natural sources of radiation in the Marshall Islands and the threshold where radiation health effects could be either medically diagnosed in an individual or epidemiologically discerned in a

  15. Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays. (United States)

    Boggiano, César; Reixach, Natàlia; Pinilla, Clemencia; Blondelle, Sylvie E


    Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.

  16. Experimental Investigations of Water Quality: The Bioassay. (United States)

    Havel, John E.; And Others


    Describes a bioassay laboratory exercise designed to introduce students to both acute and chronic bioassay procedures. Reinforces ecological principles and provides opportunities for students to use knowledge learned in the classroom in a realistic and ecologically-relevant situation. Contains 11 references. (JRH)

  17. Rapid bioassay for oil-contaminated soil

    Energy Technology Data Exchange (ETDEWEB)

    Ashworth, J. [ALS Environmental, Edmonton, AB (Canada); Oosterbroek, L. [HydroQual, Calgary, AB (Canada)


    This PowerPoint presentation described a study conducted to develop a rapid bioassay for soils contaminated with oil. The bioassay method was designed for a weight of evidence (WoE) approach and eco-contact guideline derivation protocol. Microtox bioassays were conducted on cyclodextrin extracts of soil quantified by solvent extraction and gas chromatography. The method was demonstrated using straight {beta}-cyclodextrin soil extracts and activated {beta}-cyclodextrin soil extracts. An analysis of the methods showed that the activation step weakens or breaks the cyclodextrin and polycyclic hydrocarbon (PHC) inclusion complex. The released PHC became toxic to the microtox organism. Results from the bioassays were then correlated with earthworm reproduction bioassay results. tabs., figs.

  18. Susceptibility of cat fleas (Siphonaptera: Pulicidae) to fipronil and imidacloprid using adult and larval bioassays. (United States)

    Rust, M K; Vetter, R; Denholm, I; Blagburn, B; Williamson, M S; Kopp, S; Coleman, G; Hostetler, J; Davis, W; Mencke, N; Rees, R; Foit, S; Tetzner, K


    The monitoring of the susceptibility offleas to insecticides has typically been conducted by exposing adults on treated surfaces. Other methods such as topical applications of insecticides to adults and larval bioassays on treated rearing media have been developed. Unfortunately, baseline responses of susceptible strains of cat flea, Ctenocephalides felis (Bouchè), except for imidacloprid, have not been determined for all on-animal therapies and new classes of chemistry now being used. However, the relationship between adult and larval bioassays of fleas has not been previously investigated. The adult and larval bioassays of fipronil and imidacloprid were compared for both field-collected isolates and laboratory strains. Adult topical bioassays of fipronil and imidacloprid to laboratory strains and field-collected isolates demonstrated that LD50s of fipronil and imidacloprid ranged from 0.11 to 0.40 nanograms per flea and 0.02 to 0.18 nanograms per flea, respectively. Resistance ratios for fipronil and imidacloprid ranged from 0.11 to 2.21. Based on the larval bioassay published for imidacloprid, a larval bioassay was established for fipronil and reported in this article. The ranges of the LC50s of fipronil and imidacloprid in the larval rearing media were 0.07-0.16 and 0.11-0.21 ppm, respectively. Resistance ratios for adult and larval bioassays ranged from 0.11 to 2.2 and 0.58 to 1.75, respectively. Both adult and larval bioassays provided similar patterns for fipronil and imidacloprid. Although the adult bioassays permitted a more precise dosage applied, the larval bioassays allowed for testing isolates without the need to maintain on synthetic or natural hosts.

  19. Enhanced spectral sensitivity of a chip-scale photonic-crystal slow-light interferometer

    CERN Document Server

    Magaña-Loaiza, Omar S; Schulz, Sebastian A; Awan, Kashif; Upham, Jeremy; Dolgaleva, Ksenia; Boyd, Robert W


    We experimentally demonstrate that the spectral sensitivity of a Mach-Zehnder (MZ) interferometer can be enhanced through structural slow light. We observe a 20 times enhancement by placing a dispersion-engineered-slow-light photonic-crystal waveguide in one arm of a fibre-based MZ interferometer. The spectral sensitivity of the interferometer increases roughly linearly with the group index, and we have quantified the resolution in terms of the spectral density of interference fringes. These results show promise for the use of slow-light methods for developing novel tools for optical metrology and, specifically, for compact high-resolution spectrometers.

  20. A Shrinkage Estimator for Combination of Bioassays

    Institute of Scientific and Technical Information of China (English)

    Jian Xiong; D.G. Chen; Zhen-hai Yang


    A shrinkage estimator and a maximum likelihood estimator are proposed in this paper for combination of bioassays. The shrinkage estimator is obtained in closed form which incorporates prior information just on the common log relative potency after the homogeneity test for combination of bioassays is accepted. It is a practical improvement over other estimators which require iterative procedure to obtain the estimator for the relative potency. A real data is also used to show the superiorities for the newly-proposed procedures.

  1. The knockdown resistance mutation and knockdown time in Anopheles gambiae collected from Mali evaluated through a bottle bioassay and a novel insecticide-treated net bioassay. (United States)

    Fryxell, Rebecca T Trout; Seifert, Stephanie N; Lee, Yoosook; Sacko, Adama; Lanzaro, Gregory; Cornel, Anthony


    Successful malaria management in Mali includes the use of pyrethroids and insecticide-treated nets (ITNs) for mosquito control; however, management is threatened by the spread of insecticide resistance detected via the knockdown resistance (kdr) allele. In a preliminary study, we compared the knockdown times of Anopheles gambiae from Mali using a novel ITN bioassay and the World Health Organization (WHO) bottle bioassay. Additionally, the frequency and relationship between kdr genotypes, molecular forms, and pyrethroid resistance were analyzed. The S molecular form was predominant and accounted for 76% of the assayed population. Both kdr resistant alleles, West Africa resistant (kdr-w) and East Africa resistant (kdr-e), were observed. There was no significant difference in knockdown time based on kdr genotype or molecular form of individual mosquitoes, but mosquitoes in the ITN bioassay homozygous for the kdr-w allele were knocked down significantly faster than those in the WHO bottle bioassay. The ITN bioassay provides an additional indicator of insecticide efficacy because ITNs, frequently used within homes, are the most common form of vector control and malaria prevention, and the ITN bioassays can evaluate seasonal field effects.

  2. Bioassays for the determination of nitrification inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Grunditz, Camilla


    Requirements for nitrogen reduction in wastewater treatment plants were introduced in Sweden in the early 1990's. This was a governmental move to reduce the nitrogen discharges to the Baltic and Kattegat in order to prevent eutrophication. The nitrification process in wastewater treatment plants is performed by nitrifying bacteria. These are susceptible to inhibition and it is of great importance that the influent water does not contain toxic compounds. Therefore, there is a need for assays for the determination of nitrification inhibition. This thesis describes the development and applications of such bioassays. Pure cultures of Nitrosomonas sp. and Nitrobacter sp. were isolated from activated sludge of a wastewater treatment plant. These cultures were used as test organisms in the development of bioassays for nitrification inhibition measurements. The assays are based on two different principles; cell suspensions of the bacteria, performed in test tubes, and mediated amperometric biosensors with the bacteria immobilised. Ammonia oxidation and nitrite oxidation are studied separately without interference from other organisms, which makes it easier to interpret the results. The cell suspension assays were applied to samples of industrial and municipal wastewater. The Nitrosomonas and Nitrobacter assays showed to have different inhibition patterns. A large percentage of the Swedish municipal wastewater treatment plants were found to receive inhibitory influent water, but the inhibition level was generally low. Compared to an assay based on activated sludge, the screening method, the pure culture assays found more samples of influent water strongly inhibitory or stimulating. The highest correlation was found between the screening method and the Nitrosomonas assay. The Nitrobacter assay was found to be the most sensitive method. Assessment of toxicity of a number of chemical substances was studied using the biosensors, together with the cell suspension assays

  3. Bioassay-derived dioxin equivalent concentrations in gonads and livers of the Atlantic cod females from the Baltic Sea

    NARCIS (Netherlands)

    Dabrowska, H.; Murk, A.J.; Berg, van den J.H.J.


    The DR-H4IIE.Luc bioassay is based on the ability of dioxin and dioxin-like contaminants to activate the AhR and its signal transduction pathway, a mechanism through which these contaminants elicit their toxic effects. The bioassay was used to examine the total dioxin-equivalent (TEQ) toxicity in go

  4. Efficient Experimental Design Strategies in Toxicology and Bioassay

    Directory of Open Access Journals (Sweden)

    Timothy E. O'Brien


    Full Text Available Modelling in bioassay often uses linear or nonlinear logistic regression models, and relative potency is often the focus when two or more compounds are to be compared.  Estimation in these settings is typically based on likelihood methods.  Here, we focus on the 3-parameter model representation given in Finney (1978 in which the relative potency is a model parameter.  Using key matrix results and the general equivalence theorem of Kiefer & Wolfowitz (1960, this paper establishes key design properties of the optimal design for relative potency using this model.  We also highlight aspects of subset designs for the relative potency parameter and extend geometric designs to efficient design settings of bioassay.  These latter designs are thus useful for both parameter estimation and checking for goodness-of-fit.  A typical yet insightful example is provided from the field of toxicology to illustrate our findings.

  5. An overview of the PubChem BioAssay resource. (United States)

    Wang, Yanli; Bolton, Evan; Dracheva, Svetlana; Karapetyan, Karen; Shoemaker, Benjamin A; Suzek, Tugba O; Wang, Jiyao; Xiao, Jewen; Zhang, Jian; Bryant, Stephen H


    The PubChem BioAssay database ( is a public repository for biological activities of small molecules and small interfering RNAs (siRNAs) hosted by the US National Institutes of Health (NIH). It archives experimental descriptions of assays and biological test results and makes the information freely accessible to the public. A PubChem BioAssay data entry includes an assay description, a summary and detailed test results. Each assay record is linked to the molecular target, whenever possible, and is cross-referenced to other National Center for Biotechnology Information (NCBI) database records. 'Related BioAssays' are identified by examining the assay target relationship and activity profile of commonly tested compounds. A key goal of PubChem BioAssay is to make the biological activity information easily accessible through the NCBI information retrieval system-Entrez, and various web-based PubChem services. An integrated suite of data analysis tools are available to optimize the utility of the chemical structure and biological activity information within PubChem, enabling researchers to aggregate, compare and analyze biological test results contributed by multiple organizations. In this work, we describe the PubChem BioAssay database, including data model, bioassay deposition and utilities that PubChem provides for searching, downloading and analyzing the biological activity information contained therein.

  6. Plant bioassay to assess the effects of allelochemicals on the metabolome of the target species Aegilops geniculata by an NMR-based approach. (United States)

    D'Abrosca, Brigida; Scognamiglio, Monica; Fiumano, Vittorio; Esposito, Assunta; Choi, Young Hae; Verpoorte, Robert; Fiorentino, Antonio


    A metabolomic-based approach for the study of allelopathic interactions in the Mediterranean area is proposed using Aegilops geniculata Roth (Poaceae), a Mediterranean herbaceous plant, as test species. Its metabolome has been elucidated by 1D and 2D NMR experiments. Hydroponic plant cultures of A. geniculata were treated with specific compounds of known allelopathic potential: catechol, coumarin, p-coumaric acid, p-hydroxybenzoic acid, ferulic acid and juglone. The metabolic variations due to the presence of allelochemicals have been analyzed and measured. All of the compounds showed the strongest effects at the highest concentration, with coumarin and juglone as the most active compounds, causing an increase of several metabolites. The metabolome changes in test plants confirmed the allelochemicals' reported modes of action. The results demonstrated that the proposed method is a promising tool. It can be applied to plant extracts, making it possible to evidence the metabolites responsible for the activity, as well as their mechanisms of action.

  7. Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays. (United States)

    Escher, Beate I; Allinson, Mayumi; Altenburger, Rolf; Bain, Peter A; Balaguer, Patrick; Busch, Wibke; Crago, Jordan; Denslow, Nancy D; Dopp, Elke; Hilscherova, Klara; Humpage, Andrew R; Kumar, Anu; Grimaldi, Marina; Jayasinghe, B Sumith; Jarosova, Barbora; Jia, Ai; Makarov, Sergei; Maruya, Keith A; Medvedev, Alex; Mehinto, Alvine C; Mendez, Jamie E; Poulsen, Anita; Prochazka, Erik; Richard, Jessica; Schifferli, Andrea; Schlenk, Daniel; Scholz, Stefan; Shiraishi, Fujio; Snyder, Shane; Su, Guanyong; Tang, Janet Y M; van der Burg, Bart; van der Linden, Sander C; Werner, Inge; Westerheide, Sandy D; Wong, Chris K C; Yang, Min; Yeung, Bonnie H Y; Zhang, Xiaowei; Leusch, Frederic D L


    Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

  8. A yeast bioassay for direct measurement of thyroid hormone disrupting effects in water without sample extraction, concentration, or sterilization. (United States)

    Li, Jian; Ren, Shujuan; Han, Shaolun; Li, Na


    The present study introduces an improved yeast bioassay for rapid yet sensitive evaluation of thyroid hormone disruption at the level of thyroid receptor (TR) in environmental water samples. This assay does not require water sample preparation and thus requires very little hands-on time. Based on different β-galactosidase substrates, two modified bioassays, a colorimetric bioassay and a chemiluminescent bioassay, were developed. The compounds tested included the known thyroid hormone 3,3',5-triiodo-l-thyronine (T3), the specific TR antagonist amiodarone hydrochloride (AH) and phthalate esters (PAEs), which potentially disrupt thyroid hormone signaling. The EC50 values for T3 were similar to those previously obtained using a 96-well plate bioassay. TR antagonism by AH was studied in the presence of 2.5 × 10(-7)M T3, and the concentration producing 20% of the maximum effect (RIC20) for AH was 3.1 × 10(-7)M and 7.8 × 10(-9)M for the colorimetric bioassay and chemiluminescent bioassay, respectively. None of the tested PAEs induced β-galactosidase expression, but diethylhexyl phthalate, benzyl butyl phthalate and dibutyl phthalate demonstrated TR antagonism. Furthermore, water samples collected from Guanting reservoir in Beijing were evaluated. Although TR agonism was not observed, antagonism was detected in all water samples and is expressed as AH equivalents. The toxicology equivalent quantity values obtained by the chemiluminescent bioassay ranged from 21.2 ± 1.6 to 313.9 ± 28.8 μg L(-1) AH, and similar values were obtained for the colorimetric bioassay. The present study shows that the modified yeast bioassay can be used as a valuable tool for quantification of thyroid hormone disrupting effects in environmental water samples.

  9. The extended Beer-Lambert theory for ray tracing modeling of LED chip-scaled packaging application with multiple luminescence materials (United States)

    Yuan, Cadmus C. A.


    Optical ray tracing modeling applied Beer-Lambert method in the single luminescence material system to model the white light pattern from blue LED light source. This paper extends such algorithm to a mixed multiple luminescence material system by introducing the equivalent excitation and emission spectrum of individual luminescence materials. The quantum efficiency numbers of individual material and self-absorption of the multiple luminescence material system are considered as well. By this combination, researchers are able to model the luminescence characteristics of LED chip-scaled packaging (CSP), which provides simple process steps and the freedom of the luminescence material geometrical dimension. The method will be first validated by the experimental results. Afterward, a further parametric investigation has been then conducted.

  10. Strategies for Transferring Mixtures of Organic Contaminants from Aquatic Environments into Bioassays. (United States)

    Jahnke, Annika; Mayer, Philipp; Schäfer, Sabine; Witt, Gesine; Haase, Nora; Escher, Beate I


    Mixtures of organic contaminants are ubiquitous in the environment. Depending on their persistence and physicochemical properties, individual chemicals that make up the mixture partition and distribute within the environment and might then jointly elicit toxicological effects. For the assessment and monitoring of such mixtures, a variety of cell-based in vitro and low-complexity in vivo bioassays based on algae, daphnids or fish embryos are available. A very important and sometimes unrecognized challenge is how to combine sampling, extraction and dosing to transfer the mixtures from the environment into bioassays, while conserving (or re-establishing) their chemical composition at adjustable levels for concentration-effect assessment. This article outlines various strategies for quantifiable transfer from environmental samples including water, sediment, and biota into bioassays using total extraction or polymer-based passive sampling combined with either solvent spiking or passive dosing.

  11. In vitro bioassays of non-steroidal phytoestrogens. (United States)

    Markiewicz, L; Garey, J; Adlercreutz, H; Gurpide, E


    Some of the isoflavonoids present in human diet as well as in urine are expected to exert biologic effects as they have been reported to bind to estrogen receptors and to be estrogenic in other species. This report describes the in vitro assessment of estrogenic effects of isoflavonoids using human endometrial cells and tissue. The relative estrogenic potencies (EC50 values) of estradiol, 3 dietary isoflavonoids (coumestrol, genistein and daidzein) and one of their metabolites (equol), were estimated by using a recently developed multiwell plate in vitro bioassay based on the estrogen-specific enhancement of alkaline phosphatase (AlkP) activity in human endometrial adenocarcinoma cells of the Ishikawa-Var I line. The maximal AlkP activity elicited by the isoflavonoids tested was as high as that achieved with estradiol and their effects were suppressed by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. These results indicate that estradiol and the isoflavonoids exert their effects on AlkP by similar interactions with the estrogen receptor, with potencies depending on binding affinities. The estrogenic effect of equol was confirmed by another in vitro bioassay, based on the estrogen-stimulated enhancement of prostaglandin F2 alpha output by fragments of human secretory endometrium.

  12. Mineral deficiency and the use of the FETAX bioassay to study environmental teratogens. (United States)

    Garber, Eric A E


    The Frog Embryo Teratogenesis Assay: Xenopus (FETAX) bioassay has been employed extensively to screen compounds for teratogenic activity. Recent laboratory studies have indicated that low potassium concentrations retard Xenopus laevis development. The effects of varying concentrations of minerals on Xenopus laevis embryo length and development were examined to determine the utility of the FETAX bioassay in the study of environmental teratogens. Water samples collected from 18 wetlands in Minnesota and North Dakota correlated with low mineral levels, causing delayed embryonic development in the FETAX bioassay. When the concentration of sodium or potassium was teratogenic activity after 96 h of incubation. Furthermore, the length of the embryos-an indication of development-paralleled changes in mineral composition. Comparisons between different wetlands based on changes in one specific mineral were not possible due to a synergism between various minerals. If the concentration of sodium and/or potassium was or =2 ppm, extension of the FETAX bioassay to 120 h allowed organogenesis to proceed through stage 46, as required for scoring in accordance with ASTM guidelines for the FETAX bioassay. In those cases in which the concentration of sodium and/or potassium were teratogenic activity. Published in 2002 by John Wiley & Sons, Ltd.

  13. Applicability of the CALUX bioassay for screening of dioxin levels in human milk samples

    DEFF Research Database (Denmark)

    Laier, P.; Cederberg, Tommy Licht; Larsen, John Christian;


    . The results obtained with the bioassay when testing milk extracts fractionated into dioxins/furans, non-ortho PCB and mono/di-ortho PCB fractions indicated that the correlation between the bioassay and the chemical analyses depends primarily on the A receptor activity observed in the mono/di-ortho PCB......The CALUX (chemically activated luciferase expression) bioassay based on rat hepatoma (H4IIE) cells is a sensitive assay for the detection of Ah receptor agonists like 2,3,7,8-substituted chlorinated dibenzo-p-dioxins and dibenzofurans and related PCBs. In this paper, the assay was optimized...... and applied for monitoring levels of dioxins in human milk samples. Combination effects of dioxin-like compounds were evaluated by testing potential mechanisms of interaction between seven of the major dioxin-like compounds in human milk using the isobole method. Results showed that the compounds acted...

  14. Optimal fractionation and bioassay plans for isolation of synergistic chemicals: The subtractive-combination method. (United States)

    Byers, J A


    Studies of chemical ecology of an organism are founded on the isolation and identification of a semiochemical, often comprised of two or more synergistic compounds (each Synergist alone has little activity, but presented together they are bioactive). Chromatographie fractionation and bioassay methods of binary splitting, additive combination, and subtractive combination are compared for efficiency in isolating synergists. Formulas are derived for the latter two methods that calculate the expected number of bioassay tests required for isolation of from two to five synergists from biological extracts with any number of compounds, depending on the number of initial (major) Chromatographic fractions. A computer program based on the formulas demonstrates the superiority of the subtractive-combination method. Simulations with the program were used to determine the optimal number of initial fractions for the additive- and subtractive-combination methods when isolating two to five synergists from extracts of from 25 to 1200 compounds. Methods of bioassay, isolation, identification, and field testing of semiochemicals are discussed.

  15. Bioassay, isolation and studies on the mechanism of action of neurite extension factor (United States)

    Kligman, D.


    The identification and purification of molecules active in promoting neurite outgrowth requires a sensitive reproducible bioassay. A quantitative bioassay was utilized to purify a neurite extension factor (NEF) based on counting the number of phase bright neurons with processes at least equal to one cell body diameter after 20 hrs. in culture is defined, serum free medium. Using a combination of heat treatment DEAE cellulose chromatography and gel filtration, an acidic protein of M sub r = 75,000 was highly purified. Upon reduction, it yields subunits of M sub r = 37,000. Purified fractions are active half maximally at 100 ng/ml in inducing neurite outgrowth in this bioassay. Currently, monoclonal antibodies to NEF are being produced. Female Balb C mice were immunized with the antigen and fusions with mouse myeloma cells will be performed to yield hybridoma cells.

  16. Investigating the resistance of wild oat (Avena ludoviciana Durieu.) to fenoxaprop-p-ethyl by whole plant bioassay and seed bioassay. (United States)

    Kashani, Fatemeh Bena; Alizadeh, Hasan Mohammad; Zand, Eskandar


    Greenhouse and laboratory experiments were performed to evaluate the resistant of wild oat Avena luduviciana Durieu. populations to fenoxaprop-p-ethyl. Populations of A. ludoviciana were collected from different locations in Iran, showed indications of resistance to this herbicide. Whole plant assay experiments included screening tests and dose response experiments whereas; seed bioassay experiment consisted of ID50 determination and dose response experiments. Whole plant assay experiments were conducted as a randomized complete block design in four replications. The treatments were wild oat populations included FR1, FR2, FR3, FR4 (collected from Fars province), MR1, MR2, MR3 (collected from Markazi province), KS, KR1, KR2, KR3 (collected from Khuzestan province) and S (collected from location which had never been treated previously with any graminicide). Seed bioassay experiments were conducted using a randomized design with 4 replications. On the whole plant basis, resistance was found in, KR1, KR2, KR3 and FR4 and based on a seed bioassay, these populations were also resistant to fenoxaprop-p-ethyl. Resistance ratios (R/S) of resistant populations were different. Present findings also revealed that the seed bioassay could be used as a simple, comparatively rapid, inexpensive and accurate method for identifying wild oat populations resistant to Acetyl CoA carboxylase (ACCase) inhibitors.

  17. Vicia faba bioassay for environmental toxicity monitoring: A review. (United States)

    Iqbal, Munawar


    Higher plants are recognized as excellent genetic models to detect cytogenetic and mutagenic agents and are frequently used in environmental monitoring studies. Vicia faba (V. faba) bioassay have been used to study DNA damages i.e., chromosomal and nuclear aberrations induced by metallic compounds, pesticides, complex mixtures, petroleum derivates, toxins, nanoparticles and industrial effluents. The main advantages of using V. faba is its availability round the year, economical to use, easy to grow and handle; its use does not require sterile conditions, rate of cell division is fast, chromosomes are easy to score, less expensive and more sensitive as compared to other short-term tests that require pre-preparations. The V. faba test offers evaluation of different endpoints and tested agents can be classified as cytotoxic/genotoxic/mutagenic. This test also provides understanding about mechanism of action, whether the tested agent is clastogenic or aneugenic in nature. In view of advantages offered by V. faba test system, it is used extensively to assess toxic agents and has been emerged as an important bioassay for ecotoxicological studies. Based on the applications of V. faba test to assess the environmental quality, this article offers an overview of this test system and its efficiency in assessing the cytogenetic and mutagenic agents in different classes of the environmental concerns.

  18. In vitro bioassays to evaluate complex chemical mixtures in recycled water. (United States)

    Jia, Ai; Escher, Beate I; Leusch, Frederic D L; Tang, Janet Y M; Prochazka, Erik; Dong, Bingfeng; Snyder, Erin M; Snyder, Shane A


    With burgeoning population and diminishing availability of freshwater resources, the world continues to expand the use of alternative water resources for drinking, and the quality of these sources has been a great concern for the public as well as public health professionals. In vitro bioassays are increasingly being used to enable rapid, relatively inexpensive toxicity screening that can be used in conjunction with analytical chemistry data to evaluate water quality and the effectiveness of water treatment. In this study, a comprehensive bioassay battery consisting of 36 bioassays covering 18 biological endpoints was applied to screen the bioactivity of waters of varying qualities with parallel treatments. Samples include wastewater effluent, ultraviolet light (UV) and/or ozone advanced oxidation processed (AOP) recycled water, and infiltrated recycled groundwater. Based on assay sensitivity and detection frequency in the samples, several endpoints were highlighted in the battery, including assays for genotoxicity, mutagenicity, estrogenic activity, glucocorticoid activity, arylhydrocarbon receptor activity, oxidative stress response, and cytotoxicity. Attenuation of bioactivity was found to be dependent on the treatment process and bioassay endpoint. For instance, ozone technology significantly removed oxidative stress activity, while UV based technologies were most efficient for the attenuation of glucocorticoid activity. Chlorination partially attenuated genotoxicity and greatly decreased herbicidal activity, while groundwater infiltration efficiently attenuated most of the evaluated bioactivity with the exception of genotoxicity. In some cases, bioactivity (e.g., mutagenicity, genotoxicity, and arylhydrocarbon receptor) increased following water treatment, indicating that transformation products of water treatment may be a concern. Furthermore, several types of bioassays with the same endpoint were compared in this study, which could help guide the selection

  19. In-situ bioassays using caged bivalves

    Energy Technology Data Exchange (ETDEWEB)

    Salazar, M.H.; Salazar, S.M.


    It is important to make the distinction between chemical measurements to assess bioaccumulation potential versus biological measurements to assess potential bioeffects because bioaccumulation is not a bioeffect. Caging provides a unique opportunity to make synoptic measurements of each and facilitates making these measurements over space and time. Measuring bioaccumulation in resident and transplanted bivalves has probably been the most frequently used form of an in-situ bioassay because bivalves concentrate chemicals in their tissues. They are also easy to collect, cage, and measure. The authors have refined bivalve bioassay methods by minimizing the size range of test animals, making repetitive measurements of the same individuals, and standardizing test protocols for a variety of applications. They are now attempting to standardize criteria for accepting and interpreting data in the same way that laboratory bioassays have been standardized. Growth measurements can serve two purposes in this assessment strategy: (1) An integrated biological response endpoint that is easily quantifiable and with significance to the population, and (2) A means of calibrating bioaccumulation by assessing the relative health and physiological state of tissues that have accumulated the chemicals. In general, the authors have found the highest bioconcentration factors associated with the highest growth rates, the highest concentrations ({micro}g/g) of chemicals in juvenile mussels, and the highest chemical content ({micro}g/animal) in adult mussels. Without accounting for possible dilution of chemical concentrations by tissue growth or magnification through degrowth, contaminant concentrations can be misleading. Examples are provided for the Sudbury River in Massachusetts (Elliptio complanata), San Diego Bay (Mytilus galloprovincialis), and the Harbor Island Superfund Site in Puget Sound (Mytilus trossulus).

  20. A Multichannel Bioluminescence Determination Platform for Bioassays. (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi


    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.

  1. Aspartame bioassay findings portend human cancer hazards. (United States)

    Huff, James; LaDou, Joseph


    The U.S. Food and Drug Administration (FDA) should reevaluate its position on aspartame as being safe under all conditions. Animal bioassay results predict human cancer risks, and a recent animal study confirms that there is a potential aspartame risk to humans. Aspartame is produced and packaged in China for domestic use and global distribution. Japan, France, and the United States are also major producers. No study of long-term adverse occupational health effects on aspartame workers have been conducted. The FDA should consider sponsoring a prospective epidemiologic study of aspartame workers.

  2. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors. (United States)

    Rocaboy-Faquet, Emilie; Noguer, Thierry; Romdhane, Sana; Bertrand, Cédric; Dayan, Franck Emmanuel; Barthelmebs, Lise


    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.

  3. Bioassay for Toxic and Hazardous Materials. Training Manual. (United States)

    Office of Water Program Operations (EPA), Cincinnati, OH. National Training and Operational Technology Center.

    This course is intended for personnel who have an operational or administrative responsibility for the design and use of bioassay and biomonitoring, and who have no experience in conducting static bioassays. The training consists of classroom discussions, laboratory exercises and demonstrations, and demonstration and observation activities. (CO)

  4. Are bioassays useful tools to assess redox processes and biodegradation?

    DEFF Research Database (Denmark)

    Albrechtsen, Hans-Jørgen; Pedersen, Philip Grinder; Ludvigsen, L.


    sensitive hydrochemical or geochemical parameters, levels of hydrogen, and redox potential. However, all these approaches have to be evaluated against TEAP-bioassays as the most direct measure. We assessed successfully ongoing microbial-mediated redox processes by TEAP-bioassays in degradation studies...

  5. Benthic invertebrate bioassays with toxic sediment and pore water (United States)

    Giesy, John P.; Rosiu, Cornell J.; Graney, Robert L.; Henry, Mary G.


    The relative sensitivities of bioassays to determine the toxicity of sediments were investigated and three methods of making the sample dilutions required to generate dose-response relationships were compared. The assays studied were: (a) Microtox®, a 15-min assay ofPhotobacterium phosphoreum bioluminescence inhibition by pore water; (b) 48-h Daphnia magnalethality test in pore water; (c) 10-d subchronic assay of lethality to and reduction of weight gain by Chironomus tentans performed in either whole sediment or pore water; (d) 168-h acute lethality assay of Hexagenia limbata in either whole sediment or pore water. The three methods of diluting sediments were: (a) extracting pore water from the toxic location and dilution with pore water from the control station; (b) diluting whole sediment from the toxic location with control whole sediment from a reference location, then extracting pore water; and (c) diluting toxic, whole sediment with whole sediment from a reference location, then using the whole sediment in bioassays. Based on lethality, H. limbata was the most sensitive organism to the toxicity of Detroit River sediment. Lethality of D. magna in pore water was similar to that of H. limbata in whole sediment and can be used to predict effects of whole sediment toxicity to H. limbata. The concentration required to cause a 50% reduction in C. tentans growth (10-d EC50) was approximately that which caused 50% lethality of D. magna (48-h LC50) and was similar to the toxicity that restricts benthic invertebrate colonization of contaminated sediments. While the three dilution techniques gave similar results with some assays, they gave very different results in other assays. The dose-response relationships determined by the three dilution techniques would be expected to vary with sediment, toxicant and bioassay type, and the dose-response relationship derived from each technique needs to be interpreted accordingly.

  6. Plasmonically amplified fluorescence bioassay with microarray format (United States)

    Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.


    Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

  7. Modelling larval movement data from individual bioassays. (United States)

    McLellan, Chris R; Worton, Bruce J; Deasy, William; Birch, A Nicholas E


    We consider modelling the movements of larvae using individual bioassays in which data are collected at a high-frequency rate of five observations per second. The aim is to characterize the behaviour of the larvae when exposed to attractant and repellent compounds. Mixtures of diffusion processes, as well as Hidden Markov models, are proposed as models of larval movement. These models account for directed and localized movements, and successfully distinguish between the behaviour of larvae exposed to attractant and repellent compounds. A simulation study illustrates the advantage of using a Hidden Markov model rather than a simpler mixture model. Practical aspects of model estimation and inference are considered on extensive data collected in a study of novel approaches for the management of cabbage root fly.

  8. Application of bioassays in toxicological hazard, risk and impact assessments of dredged sediments. (United States)

    Schipper, C A; Rietjens, I M C M; Burgess, R M; Murk, A J


    Given the potential environmental consequences of dumped dredged harbour sediments it is vital to establish the potential risks from exposure before disposal at sea. Currently, European legislation for disposal of contaminated sediments at sea is based on chemical analysis of a limited number of well-known contaminants for which maximum acceptable concentrations, action levels (ALs), have been set. The present paper addresses the issue of the applicability of in vitro and in vivo bioassays for hazard, risk and local impact assessment of dredged polluted sediments to be disposed of at sea. It discusses how and to what extent selected bioassays can fill in the gaps left open by chemical analysis and the way in which the bioassays may contribute to the present licensing system for disposal. Three different purposes for application were distinguished: the most basic application (A) is a rapid determination of the hazard (potential toxicity) of dredged sediments which is then compared to ALs in a licensing system. As with chemical analysis on whole sediment extracts, the bioavailability of the chemicals is not taken into account. As in vitro assays with sediment extracts are not sensitive to matrix effects, a selection of specific in vitro bioassays can be suitable fast and standardized additions for the licensing system. When the outcome of (A) does not convincingly demonstrate whether the sediment is clean enough or too polluted, further bioanalysis can help the decision making process (B). More aspects of the mostly unknown complex chemical mixtures are taken into account, including the bioavailability and chronic toxicity focusing on ecologically relevant endpoints. The ecotoxicological pressure imposed by the dredged sediments can be quantified as the potentially affected fraction (PAF) based on chemical or biological analysis of levels of contaminants in sediment or biota. To validate the predicted risk, the actual impact of dumped harbour sediments on local

  9. Comprehensive integration of homogeneous bioassays via centrifugo-pneumatic cascading. (United States)

    Godino, Neus; Gorkin, Robert; Linares, Ana V; Burger, Robert; Ducrée, Jens


    This work for the first time presents the full integration and automation concept for a range of bioassays leveraged by cascading a centrifugo-pneumatic valving scheme to sequentially move several liquids through shared channel segments for multi-step sample preparation into the detection zone. This novel centrifugo-pneumatic liquid handling significantly simplifies system manufacture by obviating the need for complex surface functionalization procedures or hybrid material integration, as it is common in conventional valving methods such as capillary burst valves or sacrificial valves. Based on the centrifugo-pneumatic valving scheme, this work presents a toolkit of operational elements implementing liquid loading/transfer, metering, mixing and sedimentation in a microstructured polymer disc. As a proof of concept for the broad class of homogeneous bioassays, the full integration and automation of a colorimetric nitrate/nitrite test for the detection of clinically relevant nitric oxide (NO) in whole blood is implemented. First, 40 μL of plasma is extracted from a 100 μL sample of human blood, incubated for one hour with the enzymatic mixture (60 μL), and finally reacted with 100 μL of colorimetric (Greiss) reagents. Following just a single loading phase at the beginning of the process, all of these steps are automated through the centrifugo-pneumatic cascade with a high level of flow control and synchronization. Our system shows good correlation with controls up to 50 μM of nitrate, which adequately covers the healthy human range (4 to 45.3 μM).

  10. Evolving BioAssay Ontology (BAO): modularization, integration and applications. (United States)

    Abeyruwan, Saminda; Vempati, Uma D; Küçük-McGinty, Hande; Visser, Ubbo; Koleti, Amar; Mir, Ahsan; Sakurai, Kunie; Chung, Caty; Bittker, Joshua A; Clemons, Paul A; Brudz, Steve; Siripala, Anosha; Morales, Arturo J; Romacker, Martin; Twomey, David; Bureeva, Svetlana; Lemmon, Vance; Schürer, Stephan C


    The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project ( to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and

  11. A statistical treatment of bioassay pour fractions (United States)

    Barengoltz, Jack; Hughes, David

    A bioassay is a method for estimating the number of bacterial spores on a spacecraft surface for the purpose of demonstrating compliance with planetary protection (PP) requirements (Ref. 1). The details of the process may be seen in the appropriate PP document (e.g., for NASA, Ref. 2). In general, the surface is mechanically sampled with a damp sterile swab or wipe. The completion of the process is colony formation in a growth medium in a plate (Petri dish); the colonies are counted. Consider a set of samples from randomly selected, known areas of one spacecraft surface, for simplicity. One may calculate the mean and standard deviation of the bioburden density, which is the ratio of counts to area sampled. The standard deviation represents an estimate of the variation from place to place of the true bioburden density commingled with the precision of the individual sample counts. The accuracy of individual sample results depends on the equipment used, the collection method, and the culturing method. One aspect that greatly influences the result is the pour fraction, which is the quantity of fluid added to the plates divided by the total fluid used in extracting spores from the sampling equipment. In an analysis of a single sample’s counts due to the pour fraction, one seeks to answer the question: What is the probability that if a certain number of spores are counted with a known pour fraction, that there are an additional number of spores in the part of the rinse not poured. This is given for specific values by the binomial distribution density, where detection (of culturable spores) is success and the probability of success is the pour fraction. A special summation over the binomial distribution, equivalent to adding for all possible values of the true total number of spores, is performed. This distribution when normalized will almost yield the desired quantity. It is the probability that the additional number of spores does not exceed a certain value. Of course

  12. The OECD program to validate the rat Hershberger bioassay to screen compounds for in vivo and androgen and antiandrogen responses: Phase-2 dose-response studies (United States)

    DESIGN: The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the g...

  13. Bioassay-Directed Fractionation of Diesel and Biodiesel Emissions (United States)

    Biofuels are being developed as alternatives to petroleum-derived products, but published research is contradictory regarding the mutagenic activity of such emissions relative to those from petroleum diesel. We performed bioassay-directed fractionation and analyzed the polycyclic...

  14. PubChem BioAssay: 2017 update (United States)

    Wang, Yanli; Bryant, Stephen H.; Cheng, Tiejun; Wang, Jiyao; Gindulyte, Asta; Shoemaker, Benjamin A.; Thiessen, Paul A.; He, Siqian; Zhang, Jian


    PubChem's BioAssay database ( has served as a public repository for small-molecule and RNAi screening data since 2004 providing open access of its data content to the community. PubChem accepts data submission from worldwide researchers at academia, industry and government agencies. PubChem also collaborates with other chemical biology database stakeholders with data exchange. With over a decade's development effort, it becomes an important information resource supporting drug discovery and chemical biology research. To facilitate data discovery, PubChem is integrated with all other databases at NCBI. In this work, we provide an update for the PubChem BioAssay database describing several recent development including added sources of research data, redesigned BioAssay record page, new BioAssay classification browser and new features in the Upload system facilitating data sharing. PMID:27899599

  15. Overview of bioassays for mutagens, carcinogens, and teratogens

    Energy Technology Data Exchange (ETDEWEB)

    Dumont, J.N.


    Bioassays to determine the risk of health hazards of man-made chemical substances are reviewed. The standard approach to testing a substance is the tier system, consisting of three levels of testing that are increasingly complex, lengthy, and costly. The paper describes the biological basis of bioassays, identifies various assays for mutagens, carcinogens and teratogens, and explains the problems involved in extrapolating test data to human risk estimates. Future improvements in assay techniques are discussed. (CR)

  16. Luminescent Lanthanide Reporters for High-Sensitivity Novel Bioassays.

    Energy Technology Data Exchange (ETDEWEB)

    Anstey, Mitchell R.; Fruetel, Julia A.; Foster, Michael E.; Hayden, Carl C.; Buckley, Heather L.; Arnold, John


    Biological imaging and assay technologies rely on fluorescent organic dyes as reporters for a number of interesting targets and processes. However, limitations of organic dyes such as small Stokes shifts, spectral overlap of emission signals with native biological fluorescence background, and photobleaching have all inhibited the development of highly sensitive assays. To overcome the limitations of organic dyes for bioassays, we propose to develop lanthanide-based luminescent dyes and demonstrate them for molecular reporting applications. This relatively new family of dyes was selected for their attractive spectral and chemical properties. Luminescence is imparted by the lanthanide atom and allows for relatively simple chemical structures that can be tailored to the application. The photophysical properties offer unique features such as narrow and non-overlapping emission bands, long luminescent lifetimes, and long wavelength emission, which enable significant sensitivity improvements over organic dyes through spectral and temporal gating of the luminescent signal.Growth in this field has been hindered due to the necessary advanced synthetic chemistry techniques and access to experts in biological assay development. Our strategy for the development of a new lanthanide-based fluorescent reporter system is based on chelation of the lanthanide metal center using absorbing chromophores. Our first strategy involves "Click" chemistry to develop 3-fold symmetric chelators and the other involves use of a new class of tetrapyrrole ligands called corroles. This two-pronged approach is geared towards the optimization of chromophores to enhance light output.

  17. Comparison of laboratory batch and flow-through microcosm bioassays. (United States)

    Clément, Bernard J P; Delhaye, Hélène L; Triffault-Bouchet, Gaëlle G


    Since 1997, we have been developing a protocol for ecotoxicological bioassays in 2-L laboratory microcosms and have applied it to the study of various pollutants and ecotoxicological risk assessment scenarios in the area of urban facilities and transport infrastructures. The effects on five different organisms (micro-algae, duckweeds, daphnids, amphipods, chironomids) are assessed using biological responses such as growth, emergence (chironomids), reproduction (daphnids) and survival, with a duration of exposure of 3 weeks. This bioassay has mainly been used as a batch bioassay, i.e., the water was not renewed during the test. A flow-through microcosm bioassay has been developed recently, with the assumption that conditions for the biota should be improved, variability reduced, and the range of exposure patterns enlarged (e.g., the possibility of maintaining constant exposure in the water column). This paper compares the results obtained in batch and flow-through microcosm bioassays, using cadmium as a model toxicant. As expected, the stabilization of physico-chemical parameters, increased organism fitness and reduced variability were observed in the flow-through microcosm bioassay.

  18. Building bio-assays with magnetic particles on a digital microfluidic platform. (United States)

    Kokalj, Tadej; Pérez-Ruiz, Elena; Lammertyn, Jeroen


    Digital microfluidics (DMF) has emerged as a promising liquid handling technology for a variety of applications, demonstrating great potential both in terms of miniaturization and automation. DMF is based on the manipulation of discrete, independently controllable liquid droplets, which makes it highly reconfigurable and reprogrammable. One of its most exclusive advantages, compared to microchannel-based microfluidics, is its ability to precisely handle solid nano- and microsized objects, such as magnetic particles. Magnetic particles have become very popular in the last decade, since their high surface-to-volume ratio and the possibility to magnetically separate them from the matrix make them perfect suitable as a solid support for bio-assay development. The potential of magnetic particles in DMF-based bio-assays has been demonstrated for various applications. In this review we discuss the latest developments of magnetic particle-based DMF bio-assays with the aim to present, identify and analyze the trends in the field. We also discuss the state-of-the art of device integration, current status of commercialization and issues that still need to be addressed. With this paper we intend to stimulate researchers to exploit and unveil the potential of these exciting tools, which will shape the future of modern biochemistry, microbiology and biomedical diagnostics.

  19. Acarine attractants: Chemoreception, bioassay, chemistry and control. (United States)

    Carr, Ann L; Roe, Michael


    The Acari are of significant economic importance in crop production and human and animal health. Acaricides are essential for the control of these pests, but at the same time, the number of available pesticides is limited, especially for applications in animal production. The Acari consist of two major groups, the mites that demonstrate a wide variety of life strategies, i.e., herbivory, predation and ectoparasitism, and ticks which have evolved obligatory hematophagy. The major sites of chemoreception in the acarines are the chelicerae, palps and tarsi on the forelegs. A unifying name, the "foretarsal sensory organ" (FSO), is proposed for the first time in this review for the sensory site on the forelegs of all acarines. The FSO has multiple sensory functions including olfaction, gustation, and heat detection. Preliminary transcriptomic data in ticks suggest that chemoreception in the FSO is achieved by a different mechanism from insects. There are a variety of laboratory and field bioassay methods that have been developed for the identification and characterization of attractants but minimal techniques for electrophysiology studies. Over the past three to four decades, significant progress has been made in the chemistry and analysis of function for acarine attractants in mites and ticks. In mites, attractants include aggregation, immature female, female sex and alarm pheromones; in ticks, the attraction-aggregation-attachment, assembly and sex pheromones; in mites and ticks host kairomones and plant allomones; and in mites, fungal allomones. There are still large gaps in our knowledge of chemical communication in the acarines compared to insects, especially relative to acarine pheromones, and more so for mites than ticks. However, the use of lure-and-kill and lure-enhanced biocontrol strategies has been investigated for tick and mite control, respectively, with significant environmental advantages which warrant further study.

  20. Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products. (United States)

    Hakovirta, J; Reunanen, J; Saris, P E J


    A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp(uv) gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.

  1. Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

    Directory of Open Access Journals (Sweden)

    Sungsoo Na


    Full Text Available Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

  2. Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells. (United States)

    Sahmi, F; Nicola, E; Price, C A


    The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive β-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities.

  3. Toxicity bioassays for water from black-odor rivers in Wenzhou, China. (United States)

    DeFu, He; RuiRui, Chen; EnHui, Zhu; Na, Chen; Bo, Yang; HuaHong, Shi; MinSheng, Huang


    Following urbanization, a large number of urban rivers were contaminated and turned to black-odor rivers. The traditional approach for detecting water quality is based on chemical or physical analysis. However, biological toxicity of black-odor water has been less addressed. As two typical black-odor rivers, Jiushanwai River (JS) and Shanxia River (SX) are tributaries of Wen-Rui Tang River in Wenzhou (south of China). The eco-safety of the urban rivers was evaluated by bioassay for water toxicity in this study. Ten and 5 sampling sites were respectively set along JS and SX. Water samples were collected monthly from October 2010 to October 2011. The general physical and chemical parameters of river water were monitored. In order to investigate the ecotoxicological effects of black-odor water, the following bioassays were used: (1) Fish acute toxicity test (Danio rerio, comprehensive toxicity), (2) luminescent bacteria bioassay (Qinghaiensis vibrio, toxicity to bacteria), and (3) tropical claw embryo assay (Xenopus tropicalis, embryo toxicity). Biotoxicity of black-odor rivers water was demonstrated by D. rerio, Q. vibrio, and X. tropicalis embryos. Toxicological effects of black-odor water were respectively shown by mortality of zebrafish, and by the relative inhibitory light rate of luminescent bacteria. However, luminescent bacteria were more sensitive to inspect biotoxicity than zebrafish. In X. tropicalis embryos test, toxicological effects of black-odor water were mostly shown by embryos' survival rate and teratogenic rate. Bioassay results showed that toxicity of SX water was higher than that of JS water, especially in summer. Statistical analysis of luminescent bacteria toxicity test showed that biotoxicity of SX and JS was high in summer, but low in winter and spring. The seasonal changes of water toxicity of the black-odor river were positively correlative with changes of water temperature (p ecotoxicological risk of black-odor rivers was demonstrated in

  4. A macroalgal germling bioassay to assess biocide concentrations in marine waters. (United States)

    Girling, J A; Thomas, K V; Brooks, S J; Smith, D J; Shahsavari, E; Ball, A S


    A bioassay method using the early life stages (germlings) of macroalgae was developed to detect toxicity of anti-fouling paint biocides. A laboratory based bioassay using Ulva intestinalis and Fucus spiralis germlings was performed with 4 common anti-fouling biocides (tributyltin (TBT), Irgarol 1051, Diuron and zinc sulphate), over a range of environmentally relevant concentrations (0.0033-10 μg l(-1)). Comparison between the two species showed that germlings of U. intestinalis were better adapted for in-situ monitoring, as germlings of F. spiralis appeared to be too robust to display sufficient growth differences. The response of U. intestinalis germling growth appeared to reflect environmental biocide concentrations. Overall the developed method showed potential for the assessment of the sub-lethal effects of anti-fouling biocides on the early developmental stages of U. intestinalis.

  5. Inkjet-printed bioassays for direct reading with a multimode DVD/Blu-Ray optical drive. (United States)

    Li, Xiaochun; Shi, Maolin; Cui, Caie; Yu, Hua-Zhong


    Compact disc-based bioassays have been developed as novel point-of-care (POC) tools for various applications in chemical analysis and biomedical diagnosis. For the fabrication of assay discs, the surface patterning and sample introduction have been restricted to manual delivery that is unfavorable for on-demand high throughput medical screening. Herein, we have adapted a conventional inkjet printer to prepare bioassays on regular DVD-Rs and accomplished quantitative analysis with a multimode DVD/Blu-Ray optical drive in conjunction with free disc diagnostic software. The feasibility and accuracy of this method have been demonstrated by the quantitative analysis of inkjet-printed biotin-streptavidin binding assays on DVD, which serves as a trial system for other complex, medically relevant sandwich-format or competitive immunoassays.

  6. Simultaneous bioassays in a microfluidic channel on plugs of different magnetic particles. (United States)

    Bronzeau, Sandrine; Pamme, Nicole


    Magnetic particles coated with specific biomolecules are often used as solid supports for bioassays but conventional test tube based techniques are time consuming and labour intensive. An alternative is to work on magnetic particle plugs immobilised inside microfluidic channels. Most research so far has focussed on immobilising one type of particle to perform one type of assay. Here we demonstrate how several assays can be performed simultaneously by flushing a sample solution over several plugs of magnetic particles with different surface coatings. Within a microchannel, three plugs of magnetic particles were immobilised with external magnets. The particles featured surface coatings of glycine, streptavidin and protein A, respectively. Reagents were then flushed through the three plugs. Molecular binding occurred between matching antigens and antibodies in continuous flow and was detected by fluorescence. This first demonstration opens the door to a quicker and easier technique for simultaneous bioassays using magnetic particles.

  7. A Novel in vitro Bioassay to Explore the Repellent Effects of Compounds Against Mosquito Aedes aegypti (Diptera: Culicidae). (United States)

    Rehman, Junaid U; Tabanca, Nurhayat; Khan, Ikhlas A


    Mosquitoes are vectors for many pathogens resulting in many deaths of humans. Repellents play an important role in reducing mosquito bites and the spread of mosquito-borne diseases. Currently, Klun & Debboun (K & D) and human-arm-based bioassay systems are used to identify repellent properties of compounds, extracts, and essential oils. Risks involved with human-arm-based systems are allergic reactions and limited replicates. We are reporting an in vitro bioassay method “NCNPR repellent bioassay (NCNPR-RB)” that can closely simulate the results of the cloth patch bioassay system used to determine repellency against mosquitoes. The NCNPRRB method uses heat to attract mosquito and edible collagen sheets as an alternate to human skin. Multiple plant compounds with documented repellency were tested. DEET (N,N-diethyl-3-methylbenzamide) was used as a positive control. Treatments were prepared in EtOH and applied in dosages ranging from 0.011–1.5mg/cm2 to a 20-cm2 collagen sheet. The number of mosquitoes commencing to bite per probe was recorded visually for 1 min. The minimum effective dosage (mg/cm2) of compounds: DEET (0.021), carvacrol (0.011), thymol (0.013), undecanoic acid (0.023), thymol methyl ether (0.269), and 2-nonanone (>0.375 mg/cm2) determined in NCNPRRB were similar to those reported in literature using a cloth patch bioassay system. The NCNPR-RB can be used to screen compounds with reasonable reproducibility of the data at a faster rate than the cloth patch bioassay, which involves the use of human subjects.

  8. Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS. (United States)

    Van Wouwe, N; Windal, I; Vanderperren, H; Eppe, G; Xhrouet, C; Massart, A-C; Debacker, N; Sasse, A; Baeyens, W; De Pauw, E; Sartor, F; Van Oyen, H; Goeyens, L


    Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid-liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.

  9. Toxicity assessment through multiple endpoint bioassays in soils posing environmental risk according to regulatory screening values. (United States)

    Rodriguez-Ruiz, A; Asensio, V; Zaldibar, B; Soto, M; Marigómez, I


    Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.

  10. Toxicity of pyrrolizidine alkaloids to Spodoptera exigua using insect cell lines and injection bioassays. (United States)

    Nuringtyas, Tri R; Verpoorte, Robert; Klinkhamer, Peter G L; van Oers, Monique M; Leiss, Kirsten A


    Pyrrolizidine alkaloids (PAs) are feeding deterrents and toxic compounds to generalist herbivores. Among the PAs of Jacobaea vulgaris Gaertn, jacobine and erucifoline are the most effective against insect herbivores as indicated by correlative studies. Because little is known about the effect of jacobine and erucifoline as individual PAs, we isolated these compounds from their respective Jacobaea chemotypes. These PAs and other commercially available senecionine-like PAs, including senecionine, seneciphylline, retrorsine, and senkirkine, were tested as free base and N-oxide forms at a range of 0-70 ppm. Feeding bioassays using live insects are closer to the natural pattern but require relatively large amounts of test compounds. We, therefore, compared the toxicity of PAs using both Spodoptera exigua cell line and larval injection bioassays. Both bioassays led to similar results in the order of PA toxicity, indicating that the cell lines are a valuable tool for a first toxicity screen. Testing individual PAs, jacobine and erucifoline were the most toxic PAs, suggesting their major role in plant defense against generalist herbivores. Senkirkine and seneciphylline were less toxic than jacobine and erucifoline but more toxic than retrorsine. Senecionine was not toxic at the tested concentrations. For all toxic PAs, the free base form was more toxic than the N-oxide form. Our results demonstrate that structural variation of PAs influences their effectiveness in plant defense.

  11. Biomarkers and Bioassays for Cardiovascular Diseases: Present and Future

    Directory of Open Access Journals (Sweden)

    Derek S. Sim


    Full Text Available Stratification of cardiac patients arriving at the emergency department is now being made according to the levels of acute cardiac biomarkers (i.e. cardiac troponin (cTn or creatine kinase myocardial band (CK-MB. Ongoing efforts are undertaken in an attempt to identify and validate additional cardiac biomarkers, for example, interleukin-6, soluble CD40L, and C-reactive protein, in order to further risk stratify patients with acute coronary syndrome. Several studies have also now shown an association of platelet transcriptome and genomic single nucleotide polymorphisms with myocardial infarction by using advanced genomic tools. A number of markers, such as myeloid-related protein 14 (MRP-14, cyclooxygenase-1 (COX-1, 5-lipoxygenase activating protein (FLAP, leukotriene A4 hydrolase (LTA4H and myocyte enhancing factor 2A (MEF2A, have been linked to acute coronary syndromes, including myocardial infarction. In the future, these novel markers may pave the way toward personalized disease-prevention programs based on a person’s genomic, thrombotic and cardiovascular profiles. Current and future biomarkers and bioassays for identifying at-risk patients will be discussed in this review.

  12. Development of a feeding behavioural bioassay using the freshwater amphipod Gammarus pulex and the Multispecies Freshwater Biomonitor.

    NARCIS (Netherlands)

    Alonso, A.; Lange, de H.J.; Peeters, E.T.H.M.


    The present study reports the development of a feeding behavioural bioassay using the Multispecies Freshwater Biomonitor (MFB). This device is based on the quadruple impedance conversion technique to record online different behaviours of animals. Animal movements in the water generate specific frequ

  13. Biomarkers and bioassays for detecting dioxin-like compounds in the marine environment. (United States)

    Hahn, Mark E


    The presence of toxic chemical contaminants in some marine organisms, including those consumed by humans, is well known. Monitoring the levels of such contaminants and their geographic and temporal variability is important for assessing and maintaining the safety of seafood and the health of the marine environment. Chemical analyses are sensitive and specific, but can be expensive and provide little information on the actual or potential biological activity of the contaminants. Biologically-based assays can be used to indicate the presence and potential effects of contaminants in marine animals, and therefore, have potential for routine monitoring of the marine environment. Halogenated aromatic hydrocarbons (HAHs) such as chlorinated dioxins, dibenzofurans, and biphenyls comprise a major group of marine contaminants. The most toxic HAHs (dioxin-like compounds) act through an intracellular receptor protein, the aryl hydrocarbon receptor, which is present in humans and many, but not all, marine animals. A toxic equivalency approach based on an understanding of this mechanism provides an integrated measure of the biological potency or activity of HAH mixtures. Biomarkers measured in marine animals indicate their exposure to these chemicals in vivo. Similarly, in vitro biomarker responses measured in cell culture bioassays can be used to assess the concentration of 'dioxin equivalents' in extracts of environmental matrices. Here, I have reviewed the types and relative sensitivities of mechanistically-based, in vitro bioassays for dioxin-like compounds, including assays of receptor-binding, DNA-binding and transcriptional activation of native (CYP1A) or reporter (luciferase) genes. Examples of their use in environmental monitoring are provided. Cell culture bioassays are rapid and inexpensive, and thus have great potential for routine monitoring of marine resources, including seafood. Several such assays exist, or are being developed, for a variety of marine

  14. Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E. (United States)

    Chatla, Kamalakar; Gaunt, Patricia; Petrie-Hanson, Lora; Hohn, Claudia; Ford, Lorelei; Hanson, Larry


    Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

  15. The interpretation of disease phenotypes to identify TSE strains following murine bioassay: characterisation of classical scrapie. (United States)

    Beck, Katy E; Vickery, Christopher M; Lockey, Richard; Holder, Thomas; Thorne, Leigh; Terry, Linda A; Denyer, Margaret; Webb, Paul; Simmons, Marion M; Spiropoulos, John


    Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.

  16. In Vitro Androgen Bioassays as a Detection Method for Designer Androgens

    Directory of Open Access Journals (Sweden)

    Alison K. Heather


    Full Text Available Androgens are the class of sex steroids responsible for male sexual characteristics, including increased muscle mass and decreased fat mass. Illicit use of androgen doping can be an attractive option for those looking to enhance sporting performance and/or physical appearance. The use of in vitro bioassays to detect androgens, especially designer or proandrogens, is becoming increasingly important in combating androgen doping associated with nutritional supplements. The nutritional sports supplement market has grown rapidly throughout the past decade. Many of these supplements contain androgens, designer androgens or proandrogens. Many designer or proandrogens cannot be detected by the standard highly-sensitive screening methods such as gas chromatography-mass spectrometry because their chemical structure is unknown. However, in vitro androgen bioassays can detect designer and proandrogens as these assays are not reliant on knowing the chemical structure but instead are based on androgen receptor activation. For these reasons, it may be advantageous to use routine androgen bioassay screening of nutraceutical samples to help curb the increasing problem of androgen doping.

  17. Bioassay for investigation of auxin transport in single cell layers

    Directory of Open Access Journals (Sweden)

    Alina B. Wodzicki


    Full Text Available Auxin was collected from the cambial region of Pinus sylvestris by applying agar strips to the cut surfaces of stem sections which comprised a single layer of 2 to 4-mm long, mainly intact fusiform cells. Sections of the agar strips were either bioassayed immediately to determine their auxin content or stored for several months at -80oC, extracted with 80% MeOH and redissolved in hot agar prior to bioassay. Auxin concentrations were determined by Went's oat coleoptile test, as described by Funke, which was modified considerably to give highly reproducible results. The modifications proved essential for good replication of results and are described in detail together with the use of the bioassay to determine changes in cambial cell polarity during ageing and senescence in P. sylvestris.

  18. High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

    DEFF Research Database (Denmark)

    Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K


    BACKGROUND: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent...... distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. METHODS: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45...

  19. [Ecotoxicological bioassays on aquatic sediments: experimental problems of exposure matrices]. (United States)

    Miniero, Roberto; Dellatte, Elena; Lupi, Carlo; Di Domenico, Alessandro


    In this review a discussion on some factors influencing the exposure matrices which, in turn, influences the reliability of ecotoxicological bioassays on aquatic sediments, has been carried out. These factors include the variability induced on sediments by the sampling, storage, handling, and preparative operations. The exposure matrices-sediments in toto, interstitial water and elutriate, can be deeply modified by these actions, which alter the chemicals bioavailability and, therefore, the bioassay meaning. In order to obtain reproducible and scientifically valid data, to be used in the ecological risk assessment, all these factors need to be considered and kept under control.

  20. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay. (United States)

    Hu, Pan; Yang, Bin


    Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays.

  1. Evaluation of the utility of the lifetime mouse bioassay in the identification of cancer hazards for humans. (United States)

    Osimitz, Thomas G; Droege, Wiebke; Boobis, Alan R; Lake, Brian G


    Limited testing resources, the need to limit animal use, and the demand for better knowledge about carcinogenic hazards require that the carcinogenicity testing paradigm based on lifetime cancer bioassays in rats and mice should be as efficient and reliable as possible. We have therefore reevaluated the rodent bioassay, particularly for nongenotoxic chemicals and conducted a rigorous examination of the 710 substances listed in the Carcinogenic Potency Database (CPDB) that were tested in both mice and rats. The CPDB is a web-based database that provides access to the literature and the results of 6540 bioassays on 1547 chemicals that have been published in the general literature through 2001 and by the National Cancer Institute/National Toxicology Program through 2004. Only three chemicals (o-benzyl-p-chlorophenol, Elmiron®, p-tolylurea) were identified as unequivocally non-genotoxic, mouse non-liver carcinogens. A careful analysis showed that their carcinogenicity in mice is irrelevant for assessment of human cancer hazards. This is consistent with data showing, with a few well-known exceptions, that non-genotoxic carcinogens in rodents are considered to be non-carcinogenic to humans. As a result, we propose that the inclusion of the mouse bioassay in the standard assessment scheme for non-genotoxic chemicals is no longer necessary.

  2. Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

    Institute of Scientific and Technical Information of China (English)

    BASTON; David; S.; KHAN; Elaine; SORRENTINO; Claudio; DENISON; Michael; S.


    Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis.The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX(Chemically Activated Luciferase Expression) and CAFLUX(Chemically Activated Fluorescent Expression) bioassays,which utilize recombinant cell lines containing stably transfected dioxin(AhR) responsive firefly luciferase or enhanced green fluorescent protein(EGFP) reporter genes,respectively.While the current CALUX and CAFLUX bioassays are very sensitive,increasing their lower limit of sensitivity,magnitude of response and dynamic range for chemical detection would significantly increase their utility,particularly for those samples that contain low levels of dioxin-like HAHs(i.e.,serum) .In this study,we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.

  3. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.

    Directory of Open Access Journals (Sweden)

    Jason M Wilham

    Full Text Available A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC. The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC and amyloid seeding assay (ASA methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD giving positive responses in 50% of replicate reactions (SD(50. Brain tissue from 263K scrapie-affected hamsters gave SD(50 values of 10(11-10(12/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50 values of 10(3.5-10(5.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50 values of 10(2.0-10(2.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of

  4. Assessment of acrylamide toxicity using a battery of standardised bioassays. (United States)

    Zovko, Mira; Vidaković-Cifrek, Željka; Cvetković, Želimira; Bošnir, Jasna; Šikić, Sandra


    Acrylamide is a monomer widely used as an intermediate in the production of organic chemicals, e.g. polyacrylamides (PAMs). Since PAMs are low cost chemicals with applications in various industries and waste- and drinking water treatment, a certain amount of non-polymerised acrylamide is expected to end up in waterways. PAMs are non-toxic but acrylamide induces neurotoxic effects in humans and genotoxic, reproductive, and carcinogenic effects in laboratory animals. In order to evaluate the effect of acrylamide on freshwater organisms, bioassays were conducted on four species: algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata, duckweed Lemna minor and water flea Daphnia magna according to ISO (International Organization for Standardisation) standardised methods. This approach ensures the evaluation of acrylamide toxicity on organisms with different levels of organisation and the comparability of results, and it examines the value of using a battery of low-cost standardised bioassays in the monitoring of pollution and contamination of aquatic ecosystems. These results showed that EC50 values were lower for Desmodesmus subspicatus and Pseudokirchneriella subcapitata than for Daphnia magna and Lemna minor, which suggests an increased sensitivity of algae to acrylamide. According to the toxic unit approach, the values estimated by the Lemna minor and Daphnia magna bioassays, classify acrylamide as slightly toxic (TU=0-1; Class 1). The results obtained from algal bioassays (Desmodesmus subspicatus and Pseudokirchneriella subcapitata) revealed the toxic effect of acrylamide (TU=1-10; Class 2) on these organisms.

  5. Soil plate bioassay: an effective method to determine ecotoxicological risks. (United States)

    Boluda, R; Roca-Pérez, L; Marimón, L


    Heavy metals have become one of the most serious anthropogenic stressors for plants and other living organisms. Having efficient and feasible bioassays available to assess the ecotoxicological risks deriving from soil pollution is necessary. This work determines pollution by Cd, Co, Cr, Cu, Ni, Pb, V and Zn in two soils used for growing rice from the Albufera Natural Park in Valencia (Spain). Both were submitted to a different degree of anthropic activity, and their ecotoxicological risk was assessed by four ecotoxicity tests to compare their effectiveness: Microtox test, Zucconi test, pot bioassay (PB) and soil plate bioassay (SPB). The sensitivity of three plant species (barley, cress and lettuce) was also assessed. The results reveal a different degree of effectiveness and level of inhibition in the target organisms' growth depending on the test applied, to such an extent that the one-way analysis of variance showed significant differences only for the plate bioassay results, with considerable inhibition of root and shoot elongation in seedlings. Of the three plant species selected, lettuce was the most sensitive species to toxic effects, followed by cress and barley. Finally, the results also indicate that the SPB is an efficient, simple and economic alternative to other ecotoxicological assays to assess toxicity risks deriving from soil pollution.

  6. Microplate Bioassay for Determining Substrate Selectivity of "Candida rugosa" Lipase (United States)

    Wang, Shi-zhen; Fang, Bai-shan


    Substrate selectivity of "Candida rugosa" lipase was tested using "p"-nitrophenyl esters of increasing chain length (C[subscript 1], C[subscript 7], C[subscript 15]) using the high-throughput screening method. A fast and easy 96-well microplate bioassay was developed to help students learn and practice biotechnological specificity screen. The…

  7. Bioassays for evaluation of medical products derived from bacterial toxins. (United States)

    Sesardic, Thea


    Bioassays play central role in evaluation of biological products and those derived from bacterial toxins often rely exclusively on in vivo models for assurance of safety and potency. This chapter reviews existing regulatory approved methods designed to provide information on potency and safety of complex biological medicines with an insight into strategies considered for alternative procedures.

  8. Microbiological bioassay using Bacillus pumilus to detect tetracycline in milk. (United States)

    Tumini, Melisa; Nagel, Orlando Guillermo; Althaus, Rafael Lisandro


    The tetracyclines (TCs) are widely used in the treatment of several diseases of cattle and their residues may be present in milk. To control these residues it is necessary to have available inexpensive screening methods, user-friendly and capable of analysing a high number of samples. The purpose of this study was to design a bioassay of microbiological inhibition in microtiter plates with spores of Bacillus pumilus to detect TCs at concentrations corresponding to the Maximum Residue Limits (MRLs). Several complementary experiments were performed to design the bioassay. In the first study, we determined the concentration of spores that produce a change in the bioassay's relative absorbance in a short time period. Subsequently, we assessed the concentration of chloramphenicol required to decrease the detection limit (DL) of TCs at MRLs levels. Thereafter, specificity, DL and cross-specificity of the bioassay were estimated. The most appropriate microbiological inhibition assay had a B. pumilus concentration of 1.6 × 10(9) spores/ml, fortified with 2500 μg chloramphenicol/l (CAP) in Mueller Hinton culture medium using brilliant black and toluidine blue as redox indicator. This bioassay detected 117 μg chlortetracycline/l, 142 μg oxytetracycline/l and 105 μg tetracycline/l by means of a change in the indicator's colour in a period of 5 h. The method showed good specificity (97.9%) which decreased slightly (93.3%) in milk samples with high somatic cell counts (>250,000 cells/ml). Furthermore, other antimicrobials studied (except neomycin) must be present in milk at high concentrations (from >5 to >100 MRLs) to produce positive results in this assay, indicating a low cross specificity.

  9. Bioassay for aquatic ecosystems review and classification; Rassegna dei principali test di ecotossicologia acquatica

    Energy Technology Data Exchange (ETDEWEB)

    Sanci, Antonella; Rosa, Silvia [ENEA, Centro Ricerche Casaccia, Rome (Italy). Dipt. Ambiente


    Bioassay play a crucial role in assessing the actual or potential impacts of anthropogenic agents on the natural environment. In this technical report, literature on bioassays for aquatic ecosystems has been reviewed and classified. Problems associated with the choice and application of bioassays are discussed.

  10. BioAssay Ontology (BAO: a semantic description of bioassays and high-throughput screening results

    Directory of Open Access Journals (Sweden)

    Smith Robin P


    Full Text Available Abstract Background High-throughput screening (HTS is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges. Results We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal Conclusions After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference.

  11. Discovery of active components in herbs using chromatographic separation coupled with online bioassay. (United States)

    De-Qiang, Li; Zhao, Jing; Wu, Dong; Shao-Ping, Li


    Discovery of bioactive compounds from complex mixtures is a challenge. In past decades, several strategies were developed and implemented for rapid and effective screening and characterization of bioactive components in complex matrices. This review mainly focused on the online strategies, which integrated the separation science, mass spectrometry, and bioactivity screening in a single platform, allowing simultaneous screening and characterization of active compounds from complex matrices, especially from the herbs. The online screening methodologies, including pre-column affinity-based screening and post-column bioassay, were discussed and their applied examples were also presented to illustrate the strengths and limitations of these approaches.

  12. An adaptive nonparametric method in benchmark analysis for bioassay and environmental studies. (United States)

    Bhattacharya, Rabi; Lin, Lizhen


    We present a novel nonparametric method for bioassay and benchmark analysis in risk assessment, which averages isotonic MLEs based on disjoint subgroups of dosages. The asymptotic theory for the methodology is derived, showing that the MISEs (mean integrated squared error) of the estimates of both the dose-response curve F and its inverse F(-1) achieve the optimal rate O(N(-4/5)). Also, we compute the asymptotic distribution of the estimate ζ~p of the effective dosage ζ(p) = F(-1) (p) which is shown to have an optimally small asymptotic variance.

  13. Investigation of independence in inter-animal tumor-type occurrences within the NTP rodent-bioassay database

    Energy Technology Data Exchange (ETDEWEB)

    Bogen, K.T. [Lawrence Livermore National Lab., CA (United States); Seilkop, S. [Analytical Sciences, Inc., Durham, NC (United States)


    Statistically significant elevation in tumor incidence at multiple histologically distinct sites is occasionally observed among rodent bioassays of chemically induced carcinogenesis. If such data are to be relied on (as they have, e.g., by the US EPA) for quantitative cancer potency assessment, their proper analysis requires a knowledge of the extent to which multiple tumor-type occurrences are independent or uncorrelated within individual bioassay animals. Although difficult to assess in a statistically rigorous fashion, a few significant associations among tumor-type occurrences in rodent bioassays have been reported. However, no comprehensive studies of animal-specific tumor-type occurrences at death or sacrifice have been conducted using the extensive set of available NTP rodent-bioassay data, on which most cancer-potency assessment for environmental chemicals is currently based. This report presents the results of such an analysis conducted on behalf of the National Research Council`s Committee on Risk Assessment for Hazardous Air Pollutants. Tumor-type associations among individual animals were examined for {approximately}2500 to 3000 control and {approximately}200 to 600 treated animals using pathology data from 62 B6C3F1 mouse studies and 61 F/344N rat studies obtained from a readily available subset of the NTP carcinogenesis bioassay database. No evidence was found for any large correlation in either the onset probability or the prevalence-at-death or sacrifice of any tumor-type pair investigated in control and treated rats and niece, although a few of the small correlations present were statistically significant. Tumor-type occurrences were in most cases nearly independent, and departures from independence, where they did occur, were small. This finding is qualified in that tumor-type onset correlations were measured only indirectly, given the limited nature of the data analyzed.

  14. Fast and accurate semantic annotation of bioassays exploiting a hybrid of machine learning and user confirmation

    Directory of Open Access Journals (Sweden)

    Alex M. Clark


    Full Text Available Bioinformatics and computer aided drug design rely on the curation of a large number of protocols for biological assays that measure the ability of potential drugs to achieve a therapeutic effect. These assay protocols are generally published by scientists in the form of plain text, which needs to be more precisely annotated in order to be useful to software methods. We have developed a pragmatic approach to describing assays according to the semantic definitions of the BioAssay Ontology (BAO project, using a hybrid of machine learning based on natural language processing, and a simplified user interface designed to help scientists curate their data with minimum effort. We have carried out this work based on the premise that pure machine learning is insufficiently accurate, and that expecting scientists to find the time to annotate their protocols manually is unrealistic. By combining these approaches, we have created an effective prototype for which annotation of bioassay text within the domain of the training set can be accomplished very quickly. Well-trained annotations require single-click user approval, while annotations from outside the training set domain can be identified using the search feature of a well-designed user interface, and subsequently used to improve the underlying models. By drastically reducing the time required for scientists to annotate their assays, we can realistically advocate for semantic annotation to become a standard part of the publication process. Once even a small proportion of the public body of bioassay data is marked up, bioinformatics researchers can begin to construct sophisticated and useful searching and analysis algorithms that will provide a diverse and powerful set of tools for drug discovery researchers.

  15. An Improved Bioassay for Cytokinins Using Cucumber Cotyledons 1 (United States)

    Fletcher, R. A.; Kallidumbil, V.; Steele, P.


    The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N6-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins. PMID:16662273

  16. An improved bioassay for cytokinins using cucumber cotyledons. (United States)

    Fletcher, R A; Kallidumbil, V; Steele, P


    The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N(6)-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.

  17. The use of cultivars of Raphanus sativus for cytokinin bioassay

    Directory of Open Access Journals (Sweden)

    Dorota Kubowicz


    Full Text Available Six cultivars of radish (Raphanus sativus were tested for their usefulness in radish cytokinin bioassay by the method of Letham (1971. The best cultivar was found to be 'Sopel Lodu' which responds well to both zeatin and 2iP over a wide range of concentrations. The fresh weight of cotyledons increased at most by 71.5% (if treated with zeatin or 101.0% (if treated with 2iP compared to untreated cotyledons. This cultivar is also sensitive to the partially purified cytokinin-like fraction isolated from the pine (Pinus silvestris cambial region. The cultivar 'Sopel Lodu' is therefore proposed to be a suitable plant for cytokinin bioassays.

  18. Development of cell-based bioassay with Sf9 cells expressing TcSKR1 and TcSKR2 and differential activation by sulfated and non-sulfated SK peptides. (United States)

    Yu, Na; Swevers, Luc; Nachman, Ronald J; Smagghe, Guy


    Insect sulfakinin receptors (SKRs) are G-protein-coupled receptors (GPCRs) that interact with sulfakinins (SKs) to modulate diverse biological processes. One of the indispensable roles of SKs is in the regulation of food intake in insects. In this project we report on the development of a cell-based receptor assay system with insect Sf9 cells, expressing TcSKR1 and TcSKR2 from the red flour beetle Tribolium castaneum, a model and important pest insect in agriculture. In this system, a stable presence of the two TcSKRs was supported by Western blotting. The expressed TcSKRs were coupled to Gαs-protein upon activation and stimulated cAMP accumulation in Sf9 cells. Exposure of the transfected cell lines to sulfated SK (sSK) activated TcSKR1 at 1 nM; the EC50 of sSK to obtain 50% of receptor activation was similar for both receptors. In contrast, μM concentrations of non-sulfated SK were necessary to activate both TcSKRs. In conclusion, this cell-based TcSKR assay system is useful to screen SK-related peptides and mimetics and to better document ligand-receptor structure-activity relationships. Given the importance of SK signaling system in insects, the present study may provide new insights on the development of new methods to control pest insects.

  19. Improved bioassay for detecting autoinducer of Rhodovulum sulfidophilum (United States)

    Terada, T.; Kikuchi, Y.; Umekage, S.


    Quorum sensing is a bacterial gene regulation system that enables prompt environmental adaptation in response to cell density. Quorum sensing is driven by an extracellularly secreted chemical signal called autoinducer. Gram-negative bacteria produce one or several types of N-acylhomoserine lactone (AHL) as autoinducers. Our previous study suggests that the gram-negative marine photosynthetic bacterium Rhodovulum sulfidophilum produces AHL in the early stationary phase and plays a role in maintaining the bacterial cell aggregates called "floc". We performed conventional bioassay to identify AHL production by using Chromobacterium violaceum VIR07, which produces violet pigment (violacein) in response to AHL with side chains ranging from C10 to C18 in length. However, we were not able to observe the violacein with good reproducibility, suggesting that inhibitory chemical compounds co-existed in the AHL extract. Therefore, we improved the extraction method; the ethyl acetate-extracted AHLs were fractionated by using reverse phase TLC. By using the re-extracted AHLs for the bioassay, we observed an obvious production of violacein. This result clearly indicates that R. sulfidophilum produces AHLs with side chains ranging from C10 to C18 in length and suggests the utility of improved bioassay for AHL detection.

  20. A Statistical Treatment of Bioassay Pour Fractions (United States)

    Barengoltz, Jack; Hughes, David W.


    The binomial probability distribution is used to treat the statistics of a microbiological sample that is split into two parts, with only one part evaluated for spore count. One wishes to estimate the total number of spores in the sample based on the counts obtained from the part that is evaluated (pour fraction). Formally, the binomial distribution is recharacterized as a function of the observed counts (successes), with the total number (trials) an unknown. The pour fraction is the probability of success per spore (trial). This distribution must be renormalized in terms of the total number. Finally, the new renormalized distribution is integrated and mathematically inverted to yield the maximum estimate of the total number as a function of a desired level of confidence ( P(fraction. The extension to recovery efficiency corrections is also presented. Now the product of recovery efficiency and pour fraction may be small enough that the likely value may be much larger than the usual calculation: the number of spores divided by that product. The use of this analysis would not be limited to microbiological data.

  1. Global parameter estimation of the Cochlodinium polykrikoides model using bioassay data

    Institute of Scientific and Technical Information of China (English)

    CHO Hong-Yeon; PARK Kwang-Soon; KIM Sung


    Cochlodinium polykrikoides is a notoriously harmful algal species that inflicts severe damage on the aquacultures of the coastal seas of Korea and Japan. Information on their expected movement tracks and boundaries of influence is very useful and important for the effective establishment of a reduction plan. In general, the information is supported by a red-tide (a.k.a algal bloom) model. The performance of the model is highly dependent on the accuracy of parameters, which are the coefficients of functions approximating the biological growth and loss patterns of theC. polykrikoides. These parameters have been estimated using the bioassay data composed of growth-limiting factor and net growth rate value pairs. In the case of theC. polykrikoides, the parameters are different from each other in accordance with the used data because the bioassay data are sufficient compared to the other algal species. The parameters estimated by one specific dataset can be viewed as locally-optimized because they are adjusted only by that dataset. In cases where the other one data set is used, the estimation error might be considerable. In this study, the parameters are estimated by all available data sets without the use of only one specific data set and thus can be considered globally optimized. The cost function for the optimization is defined as the integrated mean squared estimation error, i.e., the difference between the values of the experimental and estimated rates. Based on quantitative error analysis, the root-mean squared errors of the global parameters show smaller values, approximately 25%–50%, than the values of the local parameters. In addition, bias is removed completely in the case of the globally estimated parameters. The parameter sets can be used as the reference default values of a red-tide model because they are optimal and representative. However, additional tuning of the parameters using thein-situ monitoring data is highly required. As opposed to the bioassay

  2. Comparison of Methods of Identifying Helicobacter hepaticus in B6C3F1 Mice Used in a Carcinogenesis Bioassay


    Fox, James G.; MacGregor, Judith A.; Shen, Zeli; Li, Xiantang; Lewis, Robert; Dangler, Charles A.


    In a long-term rodent bioassay evaluating the carcinogenicity of triethanolamine, there was equivocal evidence of carcinogenic activity in male B6C3F1 mice, based on a marginal increase in the number of hepatocellular adenomas and hepatoblastomas. Interpretation was complicated by the presence of Helicobacter hepaticus in selected silver-stained liver sections which also had histological evidence of karyomegaly and oval cell hyperplasia. An increase in numbers of liver tumors, as evidence of ...

  3. Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately


    Guillaume Tetreau; Renaud Stalinski; Jean-Philippe David; Laurence Despres


    Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin se...

  4. Bioassays for TSH receptor autoantibodies, from FRTL-5 cells to TSH receptor–LH/CG receptor chimeras: the contribution of Leonard D. Kohn

    Directory of Open Access Journals (Sweden)

    Cesidio Giuliani


    Full Text Available Since the discovery 60 years ago of the long-acting thyroid stimulator by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH receptor autoantibodies (TRAbs in Graves’ disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves’ disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies against the TSH receptor. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves’ disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSH receptor, the Kohn laboratory constructed human TSH receptor–rat luteinizing hormone/ chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of nonthyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves’ disease. This review also describes the main contributions made by others researchers in TSH receptor molecular biology and TRAbs assay, especially with the development of highly potent monoclonal antibodies. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided.

  5. Bioassays for TSH Receptor Autoantibodies, from FRTL-5 Cells to TSH Receptor–LH/CG Receptor Chimeras: The Contribution of Leonard D. Kohn (United States)

    Giuliani, Cesidio; Saji, Motoyasu; Bucci, Ines; Napolitano, Giorgio


    Since the discovery 60 years ago of the “long-acting thyroid stimulator” by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) in Graves’ disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves’ disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies (moAbs) against the TSHR. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves’ disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSHR, the Kohn laboratory constructed human TSHR–rat luteinizing hormone/chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of non-thyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves’ disease. This review also describes the main contributions made by other researchers in TSHR molecular biology and TRAbs assay, especially with the development of highly potent moAbs. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided. PMID:27504107

  6. Bioassays for TSH Receptor Autoantibodies, from FRTL-5 Cells to TSH Receptor-LH/CG Receptor Chimeras: The Contribution of Leonard D. Kohn. (United States)

    Giuliani, Cesidio; Saji, Motoyasu; Bucci, Ines; Napolitano, Giorgio


    Since the discovery 60 years ago of the "long-acting thyroid stimulator" by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) in Graves' disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves' disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies (moAbs) against the TSHR. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves' disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSHR, the Kohn laboratory constructed human TSHR-rat luteinizing hormone/chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of non-thyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves' disease. This review also describes the main contributions made by other researchers in TSHR molecular biology and TRAbs assay, especially with the development of highly potent moAbs. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided.

  7. Microfabrication of MEMS alkali metal vapor cells for chip-scale atomic devices%芯片级原子器件MEMS碱金属蒸气腔室制作

    Institute of Scientific and Technical Information of China (English)

    尤政; 马波; 阮勇; 陈硕; 张高飞


    提出了基于两步低温阳极键合工艺的碱金属蒸气腔室制作方法,用于实现原子钟、原子磁力计及原子陀螺仪等器件的芯片级集成.由微机电系统(MEMS)体硅工艺制备了腔室结构.首先采用标准工艺将刻蚀有腔室的硅圆片与Pyrex玻璃阳极键合成预成型腔室,然后引入氮缓冲气体和由惰性石蜡包覆的微量碱金属铷或铯.通过两步阳极键合来密封腔室,键合温度低于石蜡燃点198℃.第一步键合预封装腔室,键合电压小于缓冲气体的击穿电压.第二步键合在大气氛围中进行,电压增至1 200 V来增强封装质量.通过高功率激光器局部加热释放碱金属,同时在腔壁上形成均匀的石蜡镀层以延长极化原子寿命.本文实现了160℃的低温阳极键合封装,键合率达到95%以上.封装的碱金属铷释放后仍具有金属光泽,实现的最小双腔室体积为6.5 mm×4.5 mm×2 mm.铷的吸收光谱表明铷有效地封装在腔室中,证明两步低温阳极键合工艺制作碱金属蒸气腔室是可行的.%This paper reported on the microfabrication of alkali metal vapor cells based on the two-step low temperature anodic bonding for the chip-scale integration of atomic clock,atomic magnetometer,atomic gyroscope and other atomic devices.Cell structures were fabricated by Micro-electromechanical System (MEMS) bulk silicon process,and the etched silicon with cells was firstly bonded to Pyrex glass to fabricate preformed chambers by the standard anodic bonding process.Then,nitrogen buffer gas and micro-scale alkali metal (rubidium or cesium) were introduced into the preformed cells.The two-step anodic bonding process was used to seal the cells at a temperature lower than the paraffin flash point (198 ℃).In the first step,bonding voltage was lower than the breakdown voltage of nitrogen buffer gas to pre-seal the cells.In the second step,the bonding was in air atmosphere,and the bonding voltage increased up to 1

  8. Addressing the recovery of feeding rates in post-exposure feeding bioassays: Cyathura carinata as a case study

    Energy Technology Data Exchange (ETDEWEB)

    Pais-Costa, Antonia Juliana [IMAR—Institute of Marine Research, MARE—Marine and Environmental Sciences Centre, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal); Acevedo, Pelayo [SaBio IREC, Instituto de Investigación en Recursos Cinegéticos (UCLM-CSIC-JCCM), Ciudad Real 13005 (Spain); Marques, João Carlos [IMAR—Institute of Marine Research, MARE—Marine and Environmental Sciences Centre, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal); Martinez-Haro, Mónica, E-mail: [IMAR—Institute of Marine Research, MARE—Marine and Environmental Sciences Centre, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal)


    Post-exposure bioassays are used in environmental assessment as a cost-effective tool, but the effects of organism's recovery after exposure to pollutant has not yet been addressed in detail. The recoveries of post-exposure feeding rates after being exposed to two sublethal concentrations of cadmium during two different exposure periods (48 h and 96 h) were evaluated under laboratory conditions using the estuarine isopod Cyathura carinata. Results showed that feeding depression was a stable endpoint up to 24 h after cadmium exposure, which is useful for ecotoxicological bioassays. - Highlights: • We studied recovery of post-exposure feeding rates 48–96 h after cadmium exposure. • The assay is based on the isopod Cyathura carinata. • Post-exposure feeding inhibition is a stable sublethal endpoint.

  9. Combined effects of estrogenic chemicals with the same mode of action using an estrogen receptor binding bioassay. (United States)

    Yang, Rong; Li, Na; Ma, Mei; Wang, Zijian


    The increasing amounts of various estrogenic chemicals coexisting in the aquatic environment may pose environmental risks. While the concept of estradiol equivalent (EEQ) has been frequently applied in studying estrogenic mixtures, few experiments have been done to prove its reliability. In this study, the reliability of EEQ and the related model concentration addition (CA) was verified based on the two-hybrid recombinant yeast bioassay when all mixture components had the same mode of action and target of action. Our results showed that the measured estrogenic effects could be well predicted by CA and EEQ for all laboratory-made mixtures using two designs, despite the varying estrogenic activity, concentration levels and ratios of the test chemicals. This suggests that when an appropriate endpoint and its relevant bioassay are chosen, CA should be valid and the application of EEQ in predicting the effect of non-equi-effect mixtures is feasible.

  10. A bioassay for the detection of benzimidazoles reveals their presence in a range of environmental samples

    Directory of Open Access Journals (Sweden)

    Terence S Crofts


    Full Text Available Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the calcofluor-bright phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities.

  11. Bioassay standardization for the detection of allelopathic compounds and environmental toxicants using lettuce

    Directory of Open Access Journals (Sweden)

    Mateus Salomão Simões


    Full Text Available The purpose of this study was to assess different experimental conditions to determine a protocol for bioassays based on seed germination and early seedling growth using lettuce (Lactuca sativa L. cv. Grand Rapids as indicator species. This protocol aims to provide support for the standardization of assays of various chemicals such as allelochemicals and environmental toxicants. The following tests were performed: time of germination, temperature, light, solution volume and Petri dish size. For each test (except for time of germination, the influence of the conditions investigated was determined by the endpoints germination percentage, germination speed index, root length, seedling fresh weight and total dry weight. The results showed that variations in the methods altered the results. It is recommended that bioassays using L. sativa L. cv. Grand Rapids be carried out for a minimum period of four days for assessments of both germination and initial growth and that the experimental conditions include a temperature of 20°C, 90-mm Petri dishes or larger, 0.1 mL cypsela solution, and continuous light or 12-hour photoperiod.

  12. Automated cytochrome c oxidase bioassay developed for ionic liquids' toxicity assessment. (United States)

    Costa, Susana P F; Martins, Bárbara S F; Pinto, Paula C A G; Saraiva, M Lúcia M F S


    A fully automated cytochrome c oxidase assay resorting to sequential injection analysis (SIA) was developed for the first time and implemented to evaluate potential toxic compounds. The bioassay was validated by evaluation of 15 ionic liquids (ILs) with distinct cationic head groups, alkyl side chains and anions. The assay was based on cytochrome c oxidase activity reduction in presence of tested compounds and quantification of inhibitor concentration required to cause 50% of enzyme activity inhibition (EC50). The obtained results demonstrated that enzyme activity was considerably inhibited by BF4 anion and ILs incorporating non-aromatic pyrrolidinium and tetrabutylphosphonium cation cores. Emim [Ac] and chol [Ac], on contrary, presented the higher EC50 values among the ILs tested. The developed automated SIA methodology is a simple and robust high-throughput screening bioassay and exhibited good repeatability in all the tested conditions (rsd<3.7%, n=10). Therefore, it is expected that due to its simplicity and low cost, the developed approach can be used as alternative to traditional screening assays for evaluation of ILs toxicity and identification of possible toxicophore structures. Additionally, the results presented in this study provide further information about ILs toxicity.

  13. A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi. (United States)

    Grovel, Olivier; Kerzaon, Isabelle; Petit, Karina; Robiou Du Pont, Thibaut; Pouchus, Yves-François


    Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.

  14. Mimicking Daphnia magna bioassay performance by an electronic tongue for urban water quality control

    Energy Technology Data Exchange (ETDEWEB)

    Kirsanov, Dmitry, E-mail: [Laboratory of Chemical Sensors, St. Petersburg State University, St. Petersburg (Russian Federation); Laboratory of Artificial Sensor Systems, ITMO University, St. Petersburg (Russian Federation); Legin, Evgeny [Laboratory of Artificial Sensor Systems, ITMO University, St. Petersburg (Russian Federation); Sensor Systems LLC, St. Petersburg (Russian Federation); Zagrebin, Anatoly; Ignatieva, Natalia; Rybakin, Vladimir [Institute of Limnology, Russian Academy of Sciences, St. Petersburg (Russian Federation); Legin, Andrey [Laboratory of Chemical Sensors, St. Petersburg State University, St. Petersburg (Russian Federation); Laboratory of Artificial Sensor Systems, ITMO University, St. Petersburg (Russian Federation)


    Highlights: • -Daphnia magna bioassay can be simulated with multisensor system. • Urban water toxicity can be predicted from potentiometric ET data. • Independent test set validation confirms statistical significance of the results. - Abstract: Toxicity is one of the key parameters of water quality in environmental monitoring. However, being evaluated as a response of living beings (as their mobility, fertility, death rate, etc.) to water quality, toxicity can only be assessed with the help of these living beings. This imposes certain restrictions on toxicity bioassay as an analytical method: biotest organisms must be properly bred, fed and kept under strictly regulated conditions and duration of tests can be quite long (up to several days), thus making the whole procedure the prerogative of the limited number of highly specialized laboratories. This report describes an original application of potentiometric multisensor system (electronic tongue) when the set of electrochemical sensors was calibrated against Daphnia magna death rate in order to perform toxicity assessment of urban waters without immediate involvement of living creatures. PRM (partial robust M) and PLS (projections on latent structures) regression models based on the data from this multisensor system allowed for prediction of toxicity of unknown water samples in terms of biotests but in the fast and simple instrumental way. Typical errors of water toxicity predictions were below 20% in terms of Daphnia death rate which can be considered as a good result taking into account the complexity of the task.

  15. Assessment of the environmental quality of coastal sediments by using a combination of in vitro bioassays. (United States)

    Pérez-Albaladejo, Elisabet; Rizzi, Juliane; Fernandes, Denise; Lille-Langøy, Roger; Karlsen, Odd André; Goksøyr, Anders; Oros, Andra; Spagnoli, Federico; Porte, Cinta


    The environmental quality of marine sediments collected in the area of influence of the Po and Danube Rivers was assessed by using a battery of bioassays based on the use of PLHC-1 cells, zebrafish-Pxr-transfected COS-7 cells, and sea bass ovarian subcellular fractions. This allowed the determination of multiple endpoints, namely, cytotoxicity, oxidative stress, induction of CYP1A, activation of zebrafish Pxr and inhibition of ovarian aromatase. Organic extracts of sediments influenced by the Danube River and collected near harbors and urban discharges showed significant cytotoxicity, CYP1A induction and inhibition of aromatase activity. An analogous response of CYP1A induction and zfPxr activation was observed, which suggests the existence of common ligands of AhR and PXR in the sediment extracts. The study highlights the usefulness of the selected bioassays to identify those sediments that could pose a risk to aquatic organisms and that require further action in order to improve their environmental quality.

  16. Assessing the detoxication efficiencies of wastewater treatment processes using a battery of bioassays/biomarkers. (United States)

    Ma, Mei; Li, Jian; Wang, Zijian


    A battery of in vitro bioassays, including a Neutral Red (NR) assay using MCF-7 cells for predicting cytotoxic chemicals, an ethoxy resorufin-O-deethylase (EROD) activity assay using H4IIE cells to check for dioxin-like chemicals, and a recombinant gene yeast assay for screening estrogenic chemicals, was conducted to assess the removal efficiencies of trace toxic chemicals by different treatment processes in the waste water treatment plant (WWTP). The effluents were extracted by solid phase extraction (SPE) and were fractionated into three fractions based on polarities. The battery of bioassays was performed for each fraction. In the battery, the toxicities of the effluents were described according to their modes of actions (MOA) or biomarkers and the properties of the toxic chemicals were categorized by their polarities and MOAs. The proposed procedure could be used as a tool to diagnose the toxic characteristics of the complicate mixture. The results showed that cytotoxic, dioxin-like and estrogenic chemicals could be detected in all samples. In the influent, cytotoxic and dioxin-like chemicals were mainly in polar fraction and estrogenic chemicals were in non-polar and moderate-polar fractions. The secondary treatment (active sludge) could remove a small amount of these toxicants. Among different types of advanced treatments, flocculation was good enough to remove most of the cytotoxic chemicals and a combination of flocculation, ozone oxidation, and post-biological treatment could eliminate most of the dioxin-like and estrogenic chemicals.

  17. Comparison of various bioassays for dioxins measurements in fuel gas, fly ash and bottom ash

    Energy Technology Data Exchange (ETDEWEB)

    Ota, S.; Kin-ichi, S. [Ministry of the Environment, Tokyo (Japan); Masatoshi, M.; Shin-ichi, S. [National Institute for Environmental Studies, Tsukuba (Japan)


    In Japan, the control standards for dioxins (PCDDs, PCDFs and Co-PCBs) in the emission gas, fly and bottom ashes from waste incinerators have been defined in the Law Concerning Special Measures against Dioxins (Dioxins Law). Based on the Dioxins law, an installation personnel of waste incinerators of specified facilities shall measure dioxins in the emission gas and fly and bottom ashes more than once every year followed by reporting the results to their prefectural governor. The present regulating procedure has been set to use high-resolution gas chromatography/ high-resolution mass spectrometry (HRGC/HRMS, hereafter GC/MS) systems to determine dioxin-concentrations. However, the GC/MS measurements are often money- and timeconsuming, since they need complicated steps for sample preparation, expensive equipments and highly skilled technicians. Therefore, it is of high priority to develop rapid and economical alternative methods to measure dioxins. Recently, various assays using biological reactions have drawn a high degree of attention as a candidate for alternative measurement methods of dioxins. During the past decade several studies demonstrated the utility of a chemical (GC/MS) and biological (bioassays/biomarkers) control of waste thermal recycling processes like pyrolysis or incineration treatment. In this paper, we report the results of our recent examinations on the possibility to apply various bioassays to supplementary methods for the present procedure.

  18. Sensitivity of different aquatic bioassays in the assessment of a new natural formicide. (United States)

    Burga-Perez, Karen F; Toumi, Hela; Cotelle, Sylvie; Ferard, Jean-François; Radetski, Claudemir M


    Agrochemicals have the potential to cause deleterious effects on living organisms and therefore they must be subjected to various (eco)toxicological studies and monitoring programs in order to protect human health and the environment. The aim of this study was to assess the ecotoxicity of a new natural formicide with a battery of three classical and three ecotox-kit tests. The former tests were performed with Aliivibrio fischeri bacteria (Lumistox test), the cladoceran Daphnia magna and Pseudokirchneriella subcapitata algae, and the latter with Thamnotoxkit F(TM) (Thamnocephalus platyurus), Ostracodtoxkit F® (Heterocypris incongruens) and LuminoTox (photosynthetic enzyme complexes). In the range of formicide concentrations tested (from 0.06 to 2.0 g L(-1)), the measurement endpoint values varied from 0.79 g L(-1) for the algal test to > 2 g L(-1) for the LuminoTox and Ostracodtoxkit F® tests. Hierarchical sensitivity ranking based on the no-observed effect concentration (NOEC) values established to assess the formicide ecotoxicity was as follows: algal growth inhibition test ≈ daphnid immobilization test ≈ bacterial luminescence inhibition test > Thamnotoxkit F™ > LuminoTox > Ostracodtoxkit F®. Overall, results from the battery of bioassays showed that this formicide preparation presents low ecotoxicity as compared to the aquatic ecotoxicity of presently commercialized formicides. In conclusion, classical aquatic bioassays are more sensitive than ecotox-kit tests in the assessment and monitoring of the new natural formicide.

  19. Application of alpha track registration technique for plutonium estimation in bioassay samples

    Energy Technology Data Exchange (ETDEWEB)

    Sawant, Pramilla D. [Internal Dosimetry Division, BARC, Trombay, Mumbai 400085 (India)], E-mail:; Prabhu, S.P. [Internal Dosimetry Division, BARC, Trombay, Mumbai 400085 (India); Kalsi, P.C. [Radiochemistry Division, BARC, Trombay, Mumbai 400085 (India)


    Bioassay monitoring is carried out for occupational workers handling plutonium (Pu) in nuclear facilities. In India, presently Pu estimation in bioassay samples is done by alpha spectrometry. The minimum detectable activity (MDA) of alpha spectrometry is 0.5 mBq for a counting period of 1 day. To reduce the load of sample counting on alpha spectrometry, an alternative method based on alpha track registration in solid state nuclear track detectors (SSNTDs) is developed in the present paper. For this purpose, few urine samples of normal subjects spiked with known amounts of Pu in the range of 0.5-5.5 mBq were exposed to CR-39 SSNTDs. The total number of alpha tracks seen in the CR-39 films of the sample and the standard were used to calculate the amount of Pu in the sample. The results of alpha track registration technique were also compared with that obtained by the well-established alpha spectrometry and were found to agree well within {+-}30%. The minimum amount of Pu that can be analyzed by this method is 0.18 mBq for an exposure period of 45 days.

  20. Evaluation of the response of Clibanarius africanus (Decapoda: Paguridae to crude oil in static bioassay

    Directory of Open Access Journals (Sweden)

    B.J. Oribhabor


    Full Text Available The acute toxicity of Nigeria Bonny light crude oil against hermit crab, Cliabanarius africanus of a tidal creek, Eastern Obolo, Akwa Ibom State, Nigeria was investigated in the laboratory under static bioassay. The test crude oil was found to be poorly toxic to the test organism, resulting in delayed mortality and consequent extension of the bioassay to 8 days. Based on the LC50, the toxicity of the test compound was more manifested on the 8 day than at 96 hour, with a toxicity factor showing that the test compound was approximately 12 times, more manifested against C. africanus on the 8 day than at 96 hour. Paired t-test showed that there was no significant difference between 96h LC50 (549.9 ml l-1 and 8d LC50 (45.2 ml l-1. The results of this study indicated that C. africanus is not a good early warning indicator for oil pollution but its response during spills could serve as a good indicator of adverse impact.

  1. [Determination of Escherichia coli Shiga-like toxins by means of the MTT bioassay]. (United States)

    Hörmansdorfer, S; Gareis, M; Bauer, J; Mayr, A


    Tissue culture cells' metabolism and viability are measured by the mitochondrial reduction rate of a yellow tetrazolium salt (MTT) to blue formazan crystals in the MTT-bioassay. Thus the MTT-bioassay is a standardizable and reproducible bioassay for measuring cytotoxicity or cytostimulation. It is shown that the MTT-bioassay is also very suitable for determining bacterial cytotoxins using Escherichia coli's Shiga-like toxins as example. 177 strains of E. coli, isolated from carcasses and organs of cattle, are classified biochemically and tested for cytotoxin production by means of the MTT-bioassay. One of these strains is recognized as producer of Shiga-like toxin 2. 4 Enterohemolysin-producing strains of E. coli are cultivated from a feces sample of a diarrhoeic nubian ibex and identified as Shiga-like toxin 1 producers by help of the MTT-bioassay.

  2. Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays. (United States)

    Li, Wei; Qiang, Weibing; Li, Jie; Li, Hui; Dong, Yifan; Zhao, Yaju; Xu, Danke


    Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications.

  3. Fluorescent bioassays for toxic metals in milk and yoghurt

    Directory of Open Access Journals (Sweden)

    Siddiki Mohammad Shohel


    Full Text Available Abstract Background From a human health viewpoint, contaminated milk and its products could be a source of long-term exposure to toxic metals. Simple, inexpensive, and on-site assays would enable constant monitoring of their contents. Bioassays that can measure toxic metals in milk or yoghurt might reduce the risk. For this purpose, the green fluorescent protein (GFP-tagged trans factors, ArsR-GFP and CadC-GFP, together with their cis elements were used to develop such bioassays. Results ArsR-GFP or CadC-GFP, which binds either toxic metal or DNA fragment including cis element, was directly mixed with cow’s milk or yoghurt within a neutral pH range. The fluorescence of GFP, which is reflected by the association/dissociation ratio between cis element and trans factor, significantly changed with increasing externally added As (III or Cd (II whereas smaller responses to externally added Pb (II and Zn (II were found. Preparation and dilution of whey fraction at low pH were essential to intrinsic zinc quantification using CadC-GFP. Using the extraction procedure and bioassay, intrinsic Zn (II concentrations ranging from 1.4 to 4.8 mg/l for milk brands and from 1.2 to 2.9 mg/kg for yoghurt brands were determined, which correlated to those determined using inductively coupled plasma atomic emission spectroscopy. Conclusions GFP-tagged bacterial trans factors and cis elements can work in the neutralized whole composition and diluted whey fraction of milk and yoghurt. The feature of regulatory elements is advantageous for establishment of simple and rapid assays of toxic metals in dairy products.

  4. Restructuring the syllabus for MD Pharmacology: Retrospection of bioassay

    Directory of Open Access Journals (Sweden)

    Sarita Mulkalwar


    Full Text Available Introduction: Career prospects in Pharmacology are witnessing a sea change due to fast and unanticipated development in the field of clinical research. Numerous openings exist now in academia, pharmaceutical industry, Clinical Research Organizations (CRO or as regulatory consultants, experimental pharmacologists, etc. In short, there are various options to choose from, depending on one′s interest. It′s high time we ponder now over the training programme for post-graduate students in Pharmacology. It needs to be revised keeping in mind the job prospects & uniqueness of the MD Pharmacology degree. Aim: To take suggestions of experienced pharmacologists on the present syllabus for MD Pharmacology and their opinion on continuation of Bioassay experiment which is currently an important part of it . Materials and Methods: A structured questionnaire was given to 30 experienced pharmacologists to seek their opinion on MD Pharmacology syllabus & continuation of Bioassay as a part of MD practical. Results: Out of 30 participants, 29 (96.6% did not use their knowledge of Bioassay during their 10 years of post MD career, whether in pharmaceutical industry or in academics. Only 5 of them (16.6% feel that experiment on bioassay should be continued in the current state. 76.7% of them wish it to be modified to a Dose Response Curve ( DRC . 6.71% feel that it should be totally scrapped. All the participants feel the need of revising current MD Pharmacology syllabus. Current syllabus is inclined more towards preparing good academicians but it lacks the proper training for creating good clinical research professionals. Medical writing, writing necessary documents for clinical trials including regulatory documents, writing an article for medical journals, marketing communication, product monograph and patient information of a clinical trial could be incorporated. They should be aware of the regulatory requirements for conducting studies on investigational drugs

  5. Aspirator Gun for High-Throughput Mosquito Bioassays (United States)


    surveillance of Aedes aegypti in San Juan, Puerto Rico. J Am Mosq Control Assoc 10:119–124. Dietrick EJ. 1961. An improved backpack motor fan for suction...Bioassays Author(s): Robert L. Aldridge, W. Wayne Wynn, Seth C. Britch, and Kenneth J. Linthicum Source: Journal of the American Mosquito Control ...Association, 28(1):65-68. 2012. Published By: The American Mosquito Control Association DOI: URL:

  6. An emergency bioassay method for (210)Po in urine. (United States)

    Guérin, Nicolas; Dai, Xiongxin


    A rapid method was developed to efficiently measure (210)Po in urine samples in an emergency situation. Polonium-210 in small urine samples (10 mL) was spontaneously deposited on a stainless steel disc in 1 M HCl at room temperature for 4 h in a polyethylene bottle. The metallic disc was then counted for 4 h by alpha spectrometry. The developed method allowed the preparation of large sample batch in a short time. The method meets the requirements for an emergency bioassay procedure.

  7. In vitro bioassay as a predictor of in vivo response

    Directory of Open Access Journals (Sweden)

    Gurevich Konstantin G


    Full Text Available Abstract Background There is a substantial discrepancy between in vitro and in vivo experiments. The purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results. Results For dose-response curve reaches a plateau in vitro we demonstrated that the in vivo response has only one maximum. For biphasic patterns of biological response in vitro both the bimodal and biphasic in vivo responses might be observed. Conclusion As the main result of this work we have demonstrated that in vivo responses might be predicted from dose-effect curves measured in vitro.

  8. A novel bioassay for the activity determination of therapeutic human brain natriuretic peptide (BNP.

    Directory of Open Access Journals (Sweden)

    Lei Yu

    Full Text Available BACKGROUND: Recombinant human brain natriuretic peptide (rhBNP is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A. METHODOLOGY/PRINCIPAL FINDINGS: An alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA. Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision. CONCLUSION/SIGNIFICANCE: The optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.

  9. Analytical methods in bioassay-directed investigations of mutagenicity of air particulate material. (United States)

    Marvin, Christopher H; Hewitt, L Mark


    The combination of short-term bioassays and analytical chemical techniques has been successfully used in the identification of a variety of mutagenic compounds in complex mixtures. Much of the early work in the field of bioassay-directed fractionation resulted from the development of a short-term bacterial assay employing Salmonella typhimurium; this assay is commonly known as the Ames assay. Ideally, analytical methods for assessment of mutagenicity of any environmental matrix should exhibit characteristics including high capacity, good selectivity, good analytical resolution, non-destructiveness, and reproducibility. A variety of extraction solvents have been employed in investigations of mutagenicity of air particulate; sequential combination of dichloromethane followed by methanol is most popular. Soxhlet extraction has been the most common extraction method, followed by sonication. Attempts at initial fractionation using different extraction solvents have met with limited success and highlight the need for fractionation schemes applicable to moderately polar and polar mutagenic compounds. Fractionation methods reported in the literature are reviewed according to three general schemas: (i) acid/base/neutral partitioning followed by fractionation using open-column chromatography and/or HPLC; (ii) fractionation based on normal-phase (NP) HPLC using a cyanopropyl or chemically similar stationary phase; and (iii) fractionation by open-column chromatography followed by NP-HPLC. The HPLC methods may be preparative, semi-preparative, or analytical scale. Variations based on acid/base/neutral partitioning followed by a chromatographic separation have also been employed. Other lesser-used approaches involve fractionation based on ion-exchange and thin-layer chromatographies. Although some of the methodologies used in contemporary studies of mutagenicity of air particulate do not represent significant advances in technology over the past 30 years, their simplicity, low

  10. A novel bioassay for anti-thyrotrophin receptor autoantibodies detects both thyroid-blocking and stimulating activity. (United States)

    Li, Y; Kim, J; Diana, T; Klasen, R; Olivo, P D; Kahaly, G J


    Autoantibodies to the thyrotrophin (TSH) receptor (anti-TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid-stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti-TSHR antibodies block TSH and can cause hypothyroidism. Thyroid-blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)-Luc cells was more than five-fold lower compared with the wild-type CHO-Luc cells. We tested the performance of this bioassay using a thyroid-blocking monoclonal antibody K1-70, established an assay cut-off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R(2) 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20-fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred-fold and still exhibit blocking activity above the cut-off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti-TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.

  11. BioAssay Research Database (BARD): chemical biology and probe-development enabled by structured metadata and result types. (United States)

    Howe, E A; de Souza, A; Lahr, D L; Chatwin, S; Montgomery, P; Alexander, B R; Nguyen, D-T; Cruz, Y; Stonich, D A; Walzer, G; Rose, J T; Picard, S C; Liu, Z; Rose, J N; Xiang, X; Asiedu, J; Durkin, D; Levine, J; Yang, J J; Schürer, S C; Braisted, J C; Southall, N; Southern, M R; Chung, T D Y; Brudz, S; Tanega, C; Schreiber, S L; Bittker, J A; Guha, R; Clemons, P A


    BARD, the BioAssay Research Database ( is a public database and suite of tools developed to provide access to bioassay data produced by the NIH Molecular Libraries Program (MLP). Data from 631 MLP projects were migrated to a new structured vocabulary designed to capture bioassay data in a formalized manner, with particular emphasis placed on the description of assay protocols. New data can be submitted to BARD with a user-friendly set of tools that assist in the creation of appropriately formatted datasets and assay definitions. Data published through the BARD application program interface (API) can be accessed by researchers using web-based query tools or a desktop client. Third-party developers wishing to create new tools can use the API to produce stand-alone tools or new plug-ins that can be integrated into BARD. The entire BARD suite of tools therefore supports three classes of researcher: those who wish to publish data, those who wish to mine data for testable hypotheses, and those in the developer community who wish to build tools that leverage this carefully curated chemical biology resource.

  12. Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

    Directory of Open Access Journals (Sweden)

    Guillaume Tetreau


    Full Text Available Bacillus thuringiensis subsp. israelensis (Bti is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans, an increased tolerance to Cry4Aa (3.5-fold and Cry11Aa toxins (8-fold was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

  13. Compatibility of hydroxypropyl-{beta}-cyclodextrin with algal toxicity bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Fai, Patricia Bi; Grant, Alastair [School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ (United Kingdom); Reid, Brian J. [School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ (United Kingdom)], E-mail:


    Numerous reports have indicated that hydrophobic organic compound bioaccessibility in sediment and soil can be determined by extraction using aqueous hydroxypropyl-{beta}-cyclodextrin (HPCD) solutions. This study establishes the compatibility of HPCD with Selenastrum capricornutum and assesses whether its presence influences the toxicity of reference toxicants. Algal growth inhibition (72 h) showed no significant (P > 0.05) difference at HPCD concentrations up to and including 20 mM. HPCD presence did not influence the toxicity of the inorganic reference toxicant (ZnSO{sub 4}), with IC50 values of 0.82 {mu}M and 0.85 {mu}M, in the presence and absence of HPCD (20 mM), respectively. However, HPCD presence (20 mM) reduced the toxicity of 2,4-dichlorophenol and the herbicides diuron and isoproturon. These reductions were attributed to inclusion complex formation between the toxicants and the HPCD cavity. Liberation of complexed toxicants, by sample manipulation prior to toxicity assessment, is proposed to provide a sensitive, high throughput, bioassay that reflects compound bioaccessibility. - Compatibility of the biomimetic HPCD extraction method with algal cell growth inhibition bioassays to assess toxicity of reference toxicants and environmental relevant herbicides.

  14. [Evaluation of Antilles fish ciguatoxicity by mouse and chick bioassays]. (United States)

    Pottier, I; Vernoux, J P


    Ciguatera is a common seafood poisoning in Western Atlantic and French West Indies. Ciguatera fish poisoning in the Caribbean is a public health problem. A toxicological study was carried out on 178 Caribbean fish specimens (26 species) captured off Guadeloupe and Saint Barthelemy between 1993 and 1999. The mouse bioassay and the chick feeding test were used to control fish edibility. Ciguatoxins presence was assumed when symptomatology was typical of ciguatera in mouse and chick. Fishes were classified in three groups: non toxic fish (edible), low toxic fish (not edible) and toxic fish (not edible). 75% of fishes were non toxic. Toxic fish specimens belonged to four families of high trophic level carnivores: Carangidae, Lutjanidae, Serranidae et Sphyraenidae. Percentages of toxic fishes to humans reached 55% for Caranx latus and 33% for Caranx bartholomaei and Caranx lugubris. Only a significant correlation between weight and toxicity was only found for C. latus and snappers. Small carnivorous groupers (Serranidae) were also toxic. Atoxic fish species were (a) pelagic fish (Coryphaena hippurus, Auxis thazard and Euthynnus pelamis), (b) invertebrates feeders (Malacanthus plumieri, Balistes vetula), (c) small high-risk fish or (d) fish of edible benthic fish families. Liver of four fishes (Mycteroperca venenosa, Caranx bartholomaei, Seriola rivoliana, Gymnothorax funebris) contained ciguatoxins at a significant level although their flesh was safe. This study confirms the usefulness of mouse and chick bioassays for sanitary control of fish.

  15. Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

    Directory of Open Access Journals (Sweden)

    Mason Helen D


    Full Text Available Abstract Background We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF. Methods A synthetic single-chain antibody (Tomlinson J phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4–7. The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Results Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. Conclusion This study demonstrates that the combination of

  16. Sample preparation for combined chemical analysis and bioassay application in water quality assessment

    NARCIS (Netherlands)

    Kolkman, A.; Schriks, M.; Brand, W; Bäuerlein, P.S.; van der Kooi, M.M.E.; van Doorn, R.H.; Emke, E.; Reus, A.; van der Linden, S.; de Voogt, P.; Heringa, M.B.


    The combination of in vitro bioassays and chemical screening can provide a powerful toolbox to determine biologically relevant compounds in water extracts. In this study, a sample preparation method is evaluated for the suitability for both chemical analysis and in vitro bioassays. A set of 39 chemi

  17. A Bioassay for Determining Resistance Levels in Tarnished Plant Bug Populations to Neonicotinoid Insecticides (United States)

    A laboratory bioassay was developed and used to test field populations of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), for resistance development to the neonicitinoid insecticides imidacloprid (Trimax®) and thiamethoxam (Centric®). The bioassay determined LC50 values by feeding...

  18. Combined effects of copper and food on the midge Chironomus riparius in whole sediment bioassays

    NARCIS (Netherlands)

    Haas, de E.M.; Paumen, M.L.; Koelmans, A.A.; Kraak, M.H.S.


    Effects observed in whole-sediment bioassays must be seen as the joint effect of all sediment characteristics. In whole-sediment bioassays. however. adverse effects oil test organisms are usually attributed to the presence of contaminants and effects of food are often ignored. The aim of this study

  19. Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays

    NARCIS (Netherlands)

    Brand, W.; de Jongh, C.M.; Linden, S.C.; Mennes, W.; Puijker, L.M.; van Leeuwen, C.J.; van Wezel, Annemarie; Schriks, M.; Heringa, M.B.


    To screen for hormonal activity in water samples, highly sensitive in vitro CALUX bioassays are available which allow detection of estrogenic (ERα), androgenic (AR), progestagenic (PR), and glucocorticoid (GR) activities. This paper presents trigger values for the ERα, AR, PR, and GR CALUX bioassays

  20. Ecological quality assessment of Dutch surface waters using a new bioassay with the cladoceran Chydorus sphaericus

    NARCIS (Netherlands)

    Pieters, B.J.; Bosman-Meijerman, D.; Steenbergen, E.; van den Brandhof, E.J.; van Beelen, P.; van der Grinten, E.; Verweij, W.; Kraak, M.H.S.


    Routine chemical monitoring gives insight in the presence of contaminants in surface waters, but not in their joint ecological effects. Therefore ecological water quality is assessed with bioassays. Recently, a new bioassay using the chydorid Chydorus sphaericus has been developed. Working with smal

  1. Submicron polyacrolein particles in situ embedded with upconversion nanoparticles for bioassay (United States)

    Generalova, A. N.; Kochneva, I. K.; Khaydukov, E. V.; Semchishen, V. A.; Guller, A. E.; Nechaev, A. V.; Shekhter, A. B.; Zubov, V. P.; Zvyagin, A. V.; Deyev, S. M.


    We report a new surface modification approach of upconversion nanoparticles (UCNPs) structured as inorganic hosts NaYF4 codoped with Yb3+ and Er3+ based on their encapsulation in a two-stage process of precipitation polymerization of acrolein under alkaline conditions in the presence of UCNPs. The use of tetramethylammonium hydroxide both as an initiator of acrolein polymerization and as an agent for UCNP hydrophilization made it possible to increase the polyacrolein yield up to 90%. This approach enabled the facile, lossless embedment of UCNPs into the polymer particles suitable for bioassay. These particles are readily dispersible in aqueous and physiological buffers, exhibiting excellent photoluminescence properties, chemical stability, and also allow the control of particle diameters. The feasibility of the as-produced photoluminescent polymer particles mean-sized 260 nm for in vivo optical whole-animal imaging was also demonstrated using a home-built epi-luminescence imaging system.We report a new surface modification approach of upconversion nanoparticles (UCNPs) structured as inorganic hosts NaYF4 codoped with Yb3+ and Er3+ based on their encapsulation in a two-stage process of precipitation polymerization of acrolein under alkaline conditions in the presence of UCNPs. The use of tetramethylammonium hydroxide both as an initiator of acrolein polymerization and as an agent for UCNP hydrophilization made it possible to increase the polyacrolein yield up to 90%. This approach enabled the facile, lossless embedment of UCNPs into the polymer particles suitable for bioassay. These particles are readily dispersible in aqueous and physiological buffers, exhibiting excellent photoluminescence properties, chemical stability, and also allow the control of particle diameters. The feasibility of the as-produced photoluminescent polymer particles mean-sized 260 nm for in vivo optical whole-animal imaging was also demonstrated using a home-built epi-luminescence imaging

  2. Development of An ICR Mouse Bioassay for Toxicity Evaluatition in Neurotoxic Poistioning Toxins-Ctiontaminated Shellfish

    Institute of Scientific and Technical Information of China (English)

    WONG Chun Kwan; HUNG Patricia; KAM Kai Man


    Objective To develop an ICR (female) mouse bioassay (MBA) for toxicity ctionfirmatition and evaluatition of neurotoxins (brevetoxins)-ctiontaminated shellfish. Methods Brevetoxins (BTX-B) as a causative agent of neurotoxic shellfish poistioning (NSP) under different shellfish matrices were intraperittioneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determinatition of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinatitions. Detectition rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at ctioncentratition of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit (80 μg/100 g shellfish tissues). The LD50 identified was 455 μg/kg for BTX-B under general shellfish matrices (excluding oyster matrices) dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentatition in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinatitions, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Ctionclusition The two ELISA analyses agree favorably (correlatition coefficient, r≥0.96;Student's t-tests, P>0.05) with the developed bioassay.

  3. Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays. (United States)

    Brand, Walter; de Jongh, Cindy M; van der Linden, Sander C; Mennes, Wim; Puijker, Leo M; van Leeuwen, Cornelis J; van Wezel, Annemarie P; Schriks, Merijn; Heringa, Minne B


    To screen for hormonal activity in water samples, highly sensitive in vitro CALUX bioassays are available which allow detection of estrogenic (ERα), androgenic (AR), progestagenic (PR), and glucocorticoid (GR) activities. This paper presents trigger values for the ERα, AR, PR, and GR CALUX bioassays for agonistic hormonal activities in (drinking) water, which define a level above which human health risk cannot be waived a priori and additional examination of specific endocrine activity may be warranted. The trigger values are based on 1) acceptable or tolerable daily intake (ADI/TDI) values of specific compounds, 2) pharmacokinetic factors defining their bioavailability, 3) estimations of the bioavailability of unknown compounds with equivalent hormonal activity, 4) relative endocrine potencies, and 5) physiological, and drinking water allocation factors. As a result, trigger values of 3.8ng 17β-estradiol (E2)-equivalents (eq)/L, 11ng dihydrotestosterone (DHT)-eq/L, 21ng dexamethasone (DEX)-eq/L, and 333ng Org2058-eq/L were derived. Benchmark Quotient (BQ) values were derived by dividing hormonal activity in water samples by the derived trigger using the highest concentrations detected in a recent, limited screening of Dutch water samples, and were in the order of (value) AR (0.41)>ERα (0.13)>GR (0.06)>PR (0.04). The application of trigger values derived in the present study can help to judge measured agonistic hormonal activities in water samples using the CALUX bioassays and help to decide whether further examination of specific endocrine activity followed by a subsequent safety evaluation may be warranted, or whether concentrations of such activity are of low priority with respect to health concerns in the human population. For instance, at one specific drinking water production site ERα and AR (but no GR and PR) activities were detected in drinking water, however, these levels are at least a factor 83 smaller than the respective trigger values, and

  4. Neotropical electric fishes (Gymnotiformes as model organisms for bioassays

    Directory of Open Access Journals (Sweden)

    Milena Ferreira


    Full Text Available Electric fishes (Gymnotiformes inhabit Central and South America and form a relatively large group with more than 200 species. Besides a taxonomic challenge due to their still unresolved systematic, wide distribution and the variety of habitats they occupy, these fishes have been intensively studied due to their peculiar use of bioelectricity for electrolocation and communication. Conventional analysis of cells, tissues and organs have been complemented with the studies on the electric organ discharges of these fishes. This review compiles the results of 13 bioassays developed during the last 50 years, which used the quickness, low costs and functionality of the bioelectric data collection of Gymnotiformes to evaluate the effects of environmental contaminants and neuroactive drugs.

  5. Bioassay and radioimmunoassay of lactogens in sera from children. (United States)

    Gout, P W; Tze, W J; Rennie, P S; Bruchovsky, N


    Lactogen levels in sera from children have been determined using the Nb 2 lymphoma cell bioassay (BA) and conventional radioimmunoassay (RIA). Assays were done on samples obtained under basal conditions and after pituitary stimulation induced by insulin or arginine administration. There was a close correspondence between BA and RIA results (r = 0.94; n = 43). The average ratio of the BA and RIA estimates of the lactogen levels (BA/RIA) was 0.86 +/- 0.13 (mean +/- SD) and 0.82 +/- 0.17 for basal and stimulated conditions, respectively. The increased secretion of lactogens after pituitary stimulation was not found to be associated with a change in the BA/RIA ratio.

  6. Neotropical electric fishes (Gymnotiformes) as model organisms for bioassays

    Institute of Scientific and Technical Information of China (English)

    Milena Ferreira; Isac Silva de Jesus; Eliana Feldberg; JoséAntônioAlves-Gomes


    Electric fishes (Gymnotiformes) inhabit Central and South America and form a relatively large group with more than 200 species. Besides a taxonomic challenge due to their still unresolved systematic, wide distribution and the variety of habitats they occupy, these fishes have been intensively studied due to their peculiar use of bioelectricity for electrolocation and communication. Conventional analysis of cells, tissues and organs have been complemented with the studies on the electric organ discharges of these fishes. This review compiles the results of 13 bioassays developed during the last 50 years, which used the quickness, low costs and functionality of the bioelectric data collection of Gymnotiformes to evaluate the effects of environmental contaminants and neuroactive drugs.

  7. Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

    Energy Technology Data Exchange (ETDEWEB)

    Boyd, E.M. [Inst. of Terrestrial Ecology, Huntingdon (United Kingdom)]|[Univ. of Aberdeen (United Kingdom). Dept. of Plant and Soil Science; Meharg, A.A.; Wright, J. [Inst. of Terrestrial Ecology, Huntingdon (United Kingdom); Killham, K. [Univ. of Aberdeen (United Kingdom). Dept. of Plant and Soil Science


    A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, river waters, and ground waters. The assay utilized decline in luminescence of lux-marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

  8. Synthetic grape volatiles attract mated Lobesia botrana females in laboratory and field bioassays. (United States)

    Anfora, Gianfranco; Tasin, Marco; De Cristofaro, Antonio; Ioriatti, Claudio; Lucchi, Andrea


    In laboratory experiments, we identified and quantified volatiles emitted by inflorescences and berries of two grape varieties (Trebbiano and Sangiovese) and examined the effects of the volatiles on oviposition by the grapevine moth Lobesia botrana. Compared to Trebbiano, Sangiovese is relatively more susceptible to L. botrana infestations under natural conditions. Chemical and electrophysiological analysis indicated only quantitative differences between the volatiles released by the two varieties. In a dual-choice oviposition bioassay based only on volatile cues, females did not show any preference between the two varieties. The six major components of the odor profiles that were GC-EAD-active to female antennae included: limonene, 4,8-dimethyl-1,(E)-3,7-nonatriene, (+/-)-linalool, (E)-caryophyllene, (E,E)-alpha-farnesene, and methyl salicylate. At the beginning of the berry touch phenological stage, their proportions were about 10:0.6:0.4:0.5:0.9:0.6 in Trebbiano and 10:1:0.4:1.5:0.4:0.3 in Sangiovese. A six-component synthetic lure (with the proportion 10:1:1:1:1:1, which approximated the ratio of components released by both varieties) was used in further laboratory oviposition bioassays. Depending on its dosage, the synthetic lure either attracted or repelled oviposition. L. botrana females laid significantly more eggs in the presence of either the grape bunches or the synthetic lure at the attractive dosage. In a release-capture experiment conducted in a field cage that covered two grapevine rows, the synthetic lure was more attractive than a grape cluster or a blank control, and it stimulated oviposition on the vegetation near the lure. The results indicate that L. botrana uses olfactory cues to select oviposition sites and that an artificial lure, containing the major volatiles released by two grape varieties, may be useful in monitoring female activity in the field.

  9. Quantitative evaluation of besifloxacin ophthalmic suspension by HPLC, application to bioassay method and cytotoxicity studies. (United States)

    Costa, Márcia C N; Barden, Amanda T; Andrade, Juliana M M; Oppe, Tércio P; Schapoval, Elfrides E S


    Besifloxacin (BSF) is a synthetic chiral fluoroquinolone developed for the topical treatment of ophthalmic infections. The present study reports the development and validation of a microbiological assay, applying the cylinder-plate method, for determination of BSF in ophthalmic suspension. To assess this methodology, the development and validation of the method was performed for the quantification of BSF by high performance liquid chromatography (HPLC). The HPLC method showed specificity, linearity in the range of 20-80 µg mL(-1) (r=0.9998), precision, accuracy and robustness. The microbiological method is based on the inhibitory effect of BSF upon the strain of Staphylococcus epidermidis ATCC 12228 used as a test microorganism. The bioassay validation method yielded excellent results and included linearity, precision, accuracy, robustness and selectivity. The assay results were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9974) in the range of 0.5-2.0 µg mL(-1), precise (inter-assay: RSD=0.84), accurate (101.4%), specific and robust. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two methods. These results confirm that the proposed microbiological method can be used as routine analysis for the quantitative determination of BSF in an ophthalmic suspension. A preliminary stability study during the HPLC validation was performed and demonstrated that BSF is unstable under UV conditions. The photodegradation kinetics of BSF in water showed a first-order reaction for the drug product (ophthalmic suspension) and a second-order reaction for the reference standard (RS) under UVA light. UVA degraded samples of BSF were also studied in order to determine the preliminary in vitro cytotoxicity against mononuclear cells. The results indicated that BSF does not alter

  10. Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Lors, Christine [Universite Lille Nord de France, 1bis rue Georges Lefevre, 59044 Lille Cedex (France); Ecole des Mines de Douai, LGCgE-MPE-GCE, 941 rue Charles-Bourseul, 59500 Douai (France); Centre National de Recherche sur les Sites et Sols Pollues, 930 Boulevard Lahure, BP 537, 59505 Douai Cedex (France); Ponge, Jean-Francois, E-mail: [Museum National d' Histoire Naturelle, Departement Ecologie et Gestion de la Biodiversite, CNRS UMR 7179, 4 Avenue du Petit-Chateau, 91800 Brunoy (France); Martinez Aldaya, Maite [Museum National d' Histoire Naturelle, Departement Ecologie et Gestion de la Biodiversite, CNRS UMR 7179, 4 Avenue du Petit-Chateau, 91800 Brunoy (France); Damidot, Denis [Universite Lille Nord de France, 1bis rue Georges Lefevre, 59044 Lille Cedex (France); Ecole des Mines de Douai, LGCgE-MPE-GCE, 941 rue Charles-Bourseul, 59500 Douai (France)


    Bioassays on aqueous and solid phases of contaminated soils were compared, belonging to a wide array of trophic and response levels and using ecoscores for evaluating ecotoxicological and genotoxicological endpoints. The method was applied to four coke factory soils contaminated mainly with PAHs, but also to a lesser extent by heavy metals and cyanides. Aquatic bioassays do not differ from terrestrial bioassays when scaling soils according to toxicity but they are complementary from the viewpoint of ecological relevance. Both aquatic and terrestrial endpoints are strongly correlated with concentrations of 3-ring PAHs. This evaluation procedure allows us to propose a cost-effective battery which embraces a wide array of test organisms and response levels: it includes two rapid bioassays (Microtox) and springtail avoidance), a micronucleus test and three bioassays of a longer duration (algal growth, lettuce germination and springtail reproduction). This battery can be recommended for a cost-effective assessment of polluted/remediated soils. - Highlights: > Comparison of liquid- and solid-phase bioassays on contaminated soils, using ecoscores. > Complementarity of liquid- and solid-phase bioassays for the evaluation of environmental hazards. > Proposal for a restricted battery of 5 most sensitive tests. > Use of this restricted battery for a cost-effective assessment of polluted/remediated soils. - Aqueous and solid phases of contaminated soils give similar results in terms of toxicity but are complementary for the evaluation of environmental hazards by ecoscores.

  11. Establishment of a bioassay for the toxicity evaluation and quality control of Aconitum herbs

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Yi [Integrative Medicine Center, 302 Military Hospital, Beijing 100039 (China); Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500 (China); Wang, Jia-bo, E-mail: [China Military Institute of Chinese Materia Medica, 302 Military Hospital, Beijing 100039 (China); Zhao, Yan-ling; Shan, Li-mei [Integrative Medicine Center, 302 Military Hospital, Beijing 100039 (China); Li, Bao-cai [Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500 (China); Fang, Fang; Jin, Cheng [Integrative Medicine Center, 302 Military Hospital, Beijing 100039 (China); Xiao, Xiao-he, E-mail: [Integrative Medicine Center, 302 Military Hospital, Beijing 100039 (China)


    Highlights: Black-Right-Pointing-Pointer A new bioassay was optimized to evaluate the toxicity of Aconitum herbs. Black-Right-Pointing-Pointer Characterizing total toxicity is its unique advantage over chemical analysis methods. Black-Right-Pointing-Pointer The application of this bioassay promotes the safe use of Aconitum herbs in clinic. - Abstract: Currently, no bioassay is available for evaluating the toxicity of Aconitum herbs, which are well known for their lethal cardiotoxicity and neurotoxicity. In this study, we established a bioassay to evaluate the toxicity of Aconitum herbs. Test sample and standard solutions were administered to rats by intravenous infusion to determine their minimum lethal doses (MLD). Toxic potency was calculated by comparing the MLD. The experimental conditions of the method were optimized and standardized to ensure the precision and reliability of the bioassay. The application of the standardized bioassay was then tested by analyzing 18 samples of Aconitum herbs. Additionally, three major toxic alkaloids (aconitine, mesaconitine, and hypaconitine) in Aconitum herbs were analyzed using a liquid chromatographic method, which is the current method of choice for evaluating the toxicity of Aconitum herbs. We found that for all Aconitum herbs, the total toxicity of the extract was greater than the toxicity of the three alkaloids. Therefore, these three alkaloids failed to account for the total toxicity of Aconitum herbs. Compared with individual chemical analysis methods, the chief advantage of the bioassay is that it characterizes the total toxicity of Aconitum herbs. An incorrect toxicity evaluation caused by quantitative analysis of the three alkaloids might be effectively avoided by performing this bioassay. This study revealed that the bioassay is a powerful method for the safety assessment of Aconitum herbs.

  12. Evaluation on the Joint Action Between Chlorsulfuron and Haloxyfop-R by Bioassay

    Institute of Scientific and Technical Information of China (English)


    The joint action between chlorsulfuron and haloxyfop-R was evaluated by bioassay with wheat and com.respectivly. The dose-response curve derived from wheat bioassay showed that the inhibition of haloxyfop-Rto wheat root growth wasn't affected by the increasing rate of chlorsulfuron. It indicated that chlorsulfuron had no antagonism to haloxyfop-R. Meanwhile ,the variation analysis of corn bioassay indicated that these two herbicides had joint action on inhibition to corn primary root growth. The joint action was evaluated as addis tive action by using Isobole Method. So chlorsulfuron and haloxyfop-R could be used as tank mixture.

  13. [Application of bioassay in quality control of Chinese materia medica-taking Radix Isatidis as an example]. (United States)

    Yan, Dan; Ren, Yongshen; Luo, Jiaoyang; Li, Hanbing; Feng, Xue; Xiao, Xiaohe


    Bioassay, which construct the characteristics consistents with Chinese medical science, is the core mode and methods for the quality control of Chinese materia medica. Taking the bioassay of Radix Isatidis as an example, the contribution, status and application of bioassay in the quality control of Chinese materia medica were introduced in this article, and two key issue (the selection of reference and measurement methods) in the process of establishing bioassay were also explained. This article expects to provide a reference for the development and improvement of the bioassay of Chinese materia medica in a practical manipulation level.

  14. Analyzing bioassay data using Bayesian methods -- A primer

    Energy Technology Data Exchange (ETDEWEB)

    Miller, G.; Inkret, W.C.; Schillaci, M.E.


    The classical statistics approach used in health physics for the interpretation of measurements is deficient in that it does not allow for the consideration of needle in a haystack effects, where events that are rare in a population are being detected. In fact, this is often the case in health physics measurements, and the false positive fraction is often very large using the prescriptions of classical statistics. Bayesian statistics provides an objective methodology to ensure acceptably small false positive fractions. The authors present the basic methodology and a heuristic discussion. Examples are given using numerically generated and real bioassay data (Tritium). Various analytical models are used to fit the prior probability distribution, in order to test the sensitivity to choice of model. Parametric studies show that the normalized Bayesian decision level k{sub {alpha}}-L{sub c}/{sigma}{sub 0}, where {sigma}{sub 0} is the measurement uncertainty for zero true amount, is usually in the range from 3 to 5 depending on the true positive rate. Four times {sigma}{sub 0} rather than approximately two times {sigma}{sub 0}, as in classical statistics, would often seem a better choice for the decision level.

  15. Tobacco specific N-nitrosamines: occurrence and bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, D.; Adams, J.D.; Brunnemann, K.D.; Rivenson, A.; Hecht, S.S.


    A new GC-TEA method for the analysis of tobacco-specific N-nitrosamines (TSNA) has been developed. Four TSNA have thus far been identified; these are N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoanabasine (NAB). The method is currently being applied to the development of cigarette filter-tips which will selectively remove these carcinogens from cigarette smoke. Since recent epidemiological studies have established a correlation between snuff dipping and oral cancer, we have analysed leading snuff brands for TSNA. Snuff products from Sweden, Denmark, Bavaria and the USA contained 5-106 mg/kg of the TSNA and the saliva of snuff dippers had TSNA levels of 20-890 micrograms/kg. NNN, NNK and NAB induce benign and malignant tumours of the respiratory tract of mice and rats. We have shown that NNN and NNK induce tumours in the upper respiratory tract of hamsters and that NNK is the most active carcinogen of the TSNA, also inducing adenoma and adenocarcinoma in the hamster lung. The reported chemical analyses and bioassay results support the epidemiological findings on the causal association of tobacco use and cancer in man.

  16. Antioxidant, antimicrobial and cytotoxic bioassay of Mollugo oppositifolius L

    Directory of Open Access Journals (Sweden)

    Md. Torequl Islam


    Full Text Available The present study was conducted according to the traditional uses of Mollugo oppositifolius L. by the kabiraj (traditional practioner for the treatment of infectious diseases of the ill fated and poor people of Bangladesh. For this antioxidant, antimicrobial and biolethality potentials were conducted by methanol (MOME, ethanol (MOEE and petroleum ether (MOPE extractives of the suspected species. To determine the antioxidant activity the DPPH inhibition method was used. For antimicrobial test, antibacterial and antifungal sensitivities were performed by disc diffusion method and serial tube dilution method was carried out to determine the minimum inhibitory concentrations on some human pathogenic bacteria and fungi. For cytotoxicity test, brine shrimp lethality bioassay was conducted. Among the three crude extracts, MOEE produced more significant inhibition of DPPH (IC 50 ; 27 μg/ml; MOPE produced highest zone of inhibition against Bacillus subtilis (16.67 mm and Microsporum spp. (16.0 mm. On the other hand, MOME produced mild cytotoxicity as 50% and 90% mortality (LC 50 and LC 90 8.0 μg/ml and 85.12 μg/ml.

  17. Phototoxicity activity of Psoralea drupacea L. using Atremia salina bioassay system

    Directory of Open Access Journals (Sweden)

    Mohammad Ramezani


    Conclusion: The result showed that P. drupacea methanolic extract and chloroform fraction have phototoxicity in A. salina bioassay system and their toxic effect is related to phototoxic constituents such as psoralen.

  18. Estrogenic and progestagenic activities coexisting in steroidal drugs: quantitative evaluation by in vitro bioassays with human cells. (United States)

    Markiewicz, L; Gurpide, E


    The progestin-specific stimulation of alkaline phosphatase (AP) activity in cells of the T47D human breast cancer line was applied to the development of a sensitive microtiter plate bioassay for the quantitative evaluation of progestagenic and antiprogestagenic potencies of natural and synthetic compounds. Some of the steroids tested (viz. progesterone, medroxyprogesterone acetate, norethynodrel) behaved as full-agonists, capable of inducing AP activities to the same maximal levels (equal efficacy), while others (norethindrone, gestrinone, R5020, norgestrel, Org OD 14 and its 4-ene metabolite) behaved as partial agonists, eliciting lower maximal effects. Efficacy, EC50 values (concentrations at which they induce one-half of the maximal response) and "slope factors" serve to characterize agonistic effects. Relative progestagenic potencies among the full-agonists were evaluated by comparing EC50 concentrations. Several 19-nor synthetic progestins (norethynodrel, norethindrone, Org OD 14 and its 4-ene isomer, dl-norgestrel, levo-norgestrel, RU2323), but none of the tested progestins with the pregnane structure, showed intrinsic estrogenic activity, as evaluated by using a similar in vitro bioassay based on a previously reported estrogen-specific induction of AP in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. Maximal estrogenic effects of all the tested progestins with dual activity were as high as those of estradiol. However, these compounds widely varied in their EC50 values for estrogenic activity. Consequently, the in vitro bioassays can reveal differences in the ratio of progestagenic and estrogenic activities intrinsic to these compounds. The reduced capability of the partial agonists to exert progestagenic or estrogenic effects on AP expression may reflect an impeded, receptor-mediated action, a mechanism that would also account for their inhibitory effects on the induction of AP activity by full agonists. Partial progestagenic agonists

  19. Validation of a Novel Bioassay for Low-level Perchlorate Determination (United States)


    chamber using the plate reader bioassay format. 3. IC analysis for common anions showed that nitrate was present in SPE effluents at about the same...Compare Benchtop Bioassay Field Kit Results to Site’s Reference Method Results 13 3.3 Qualitative Performance Objective: Field Kit Ease of Use... Nitrate , Sulfate, Fluoride, Nitrite, Bromide, Chlorate, and Phosphate 143 Appendix I: ICPMS Characterization of Groundwater Sources and SPE Eluates

  20. Review of Bioassays for Monitoring Fate and Transport of Estrogenic Endocrine Disrupting Compounds in Water


    Campbell, Chris G.; Borglin, Sharon E.; Stringfellow, William T.; Green, F. Bailey; Grayson, Allen


    Endocrine disrupting compounds (EDCs) are recognized contaminants threatening water quality. Despite efforts in source identification, few strategies exist for characterization or treatment of this environmental pollution. Given that there are numerous EDCs that can negatively affect humans and wildlife, general screening techniques like bioassays and biosensors provide an essential rapid and intensive analysis capacity. Commonly applied bioassays include the ELISA and YES assays, but pr...

  1. A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops. (United States)

    Graser, Gerson; Walters, Frederick S


    Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet.

  2. The analysis of dose-response curve from bioassays with quantal response: Deterministic or statistical approaches? (United States)

    Mougabure-Cueto, G; Sfara, V


    Dose-response relations can be obtained from systems at any structural level of biological matter, from the molecular to the organismic level. There are two types of approaches for analyzing dose-response curves: a deterministic approach, based on the law of mass action, and a statistical approach, based on the assumed probabilities distribution of phenotypic characters. Models based on the law of mass action have been proposed to analyze dose-response relations across the entire range of biological systems. The purpose of this paper is to discuss the principles that determine the dose-response relations. Dose-response curves of simple systems are the result of chemical interactions between reacting molecules, and therefore are supported by the law of mass action. In consequence, the shape of these curves is perfectly sustained by physicochemical features. However, dose-response curves of bioassays with quantal response are not explained by the simple collision of molecules but by phenotypic variations among individuals and can be interpreted as individual tolerances. The expression of tolerance is the result of many genetic and environmental factors and thus can be considered a random variable. In consequence, the shape of its associated dose-response curve has no physicochemical bearings; instead, they are originated from random biological variations. Due to the randomness of tolerance there is no reason to use deterministic equations for its analysis; on the contrary, statistical models are the appropriate tools for analyzing these dose-response relations.

  3. A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test

    Energy Technology Data Exchange (ETDEWEB)

    Ferrao-Filho, Aloysio da S., E-mail: aloysio@ioc.fiocruz.b [Laboratorio de Avaliacao e Promocao da Saude Ambiental, Departamento de Biologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900 (Brazil); Soares, Maria Carolina S., E-mail: [Departamento de Engenharia Sanitaria e Ambiental Faculdade de Engenharia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900 (Brazil); Freitas de Magalhaes, Valeria, E-mail: valeria@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil); Azevedo, Sandra M.F.O., E-mail: sazevedo@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil)


    Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET{sub 50}) was adopted as the endpoint. Paralysis of swimming movements was observed between approx0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET{sub 50}vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays. - A new Daphnia bioassay, as an alternative to the mouse bioassay, is able to detect effects of fast-acting, potent neurotoxins in raw water.

  4. Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays. (United States)

    Dassanayake, Rohana P; Truscott, Thomas C; Zhuang, Dongyue; Schneider, David A; Madsen-Bouterse, Sally A; Young, Alan J; Stanton, James B; Davis, William C; O'Rourke, Katherine I


    Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.

  5. Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry. (United States)

    Hageneder, Simone; Bauch, Martin; Dostalek, Jakub


    This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout.

  6. Low-Level Plutonium Bioassay Measurements at the Lawrence Livermore National Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, T; Brown, T; Hickman, D; Marchetti, A; Williams, R; Kehl, S


    Plutonium-239 ({sup 239}Pu) and plutonium-240 ({sup 240}Pu) are important alpha emitting radionuclides contained in radioactive debris from nuclear weapons testing. {sup 239}Pu and {sup 240}Pu are long-lived radionuclides with half-lives of 24,400 years and 6580 years, respectively. Concerns over human exposure to plutonium stem from knowledge about the persistence of plutonium isotopes in the environment and the high relative effectiveness of alpha-radiation to cause potential harm to cells once incorporated into the human body. In vitro bioassay tests have been developed to assess uptakes of plutonium based on measured urinary excretion patterns and modeled metabolic behaviors of the absorbed radionuclides. Systemic plutonium absorbed by the deep lung or from the gastrointestinal tract after ingestion is either excreted or distributed to other organs, primarily to the liver and skeleton, where it is retained for biological half-times of around 20 and 50 years, respectively. Dose assessment and atoll rehabilitation programs in the Marshall Islands have historically given special consideration to residual concentrations of plutonium in the environment even though the predicted dose from inhalation and/or ingestion of plutonium accounts for less than 5% of the annual effective dose from exposure to fallout contamination. Scientists from the Lawrence Livermore National Laboratory (LLNL) have developed a state-of-the-art bioassay test to assess urinary excretion rates of plutonium from Marshallese populations. This new heavy-isotope measurement system is based on Accelerator Mass Spectrometry (AMS). The AMS system at LLNL far exceeds the standard measurement requirements established under the latest United States Department of Energy (DOE) regulation, 10CFR 835, for occupational monitoring of plutonium, and offers several advantages over classical as well as competing new technologies for low-level detection and measurement of plutonium isotopes. The United States

  7. Episodic acidification of small streams in the northeastern united states: Fish mortality in field bioassays (United States)

    Van Sickle, J.; Baker, J.P.; Simonin, H.A.; Baldigo, Barry P.; Kretser, W.A.; Sharpe, W.E.


    In situ bioassays were performed as part of the Episodic Response Project, to evaluate the effects of episodic stream acidification on mortality of brook trout (Salvelinus fontinalis) and forage fish species. We report the results of 122 bioassays in 13 streams of the three study regions: the Adirondack mountains of New York, the Catskill mountains of New York, and the Northern Appalachian Plateau of Pennsylvania. Bioassays during acidic episodes had significantly higher mortality than did bioassays conducted under nonacidic conditions, but there was little difference in mortality rates in bioassays experiencing acidic episodes and those experiencing acidic conditions throughout the test period. Multiple logistic regression models were used to relate bioassay mortality rates to summary statistics of time-varying stream chemistry (inorganic monomeric aluminum, calcium, pH, and dissolved organic carbon) estimated for the 20-d bioassay periods. The large suite of candidate regressors also included biological, regional, and seasonal factors, as well as several statistics summarizing various features of aluminum exposure duration and magnitude. Regressor variable selection and model assessment were complicated by multicol-linearity and overdispersion. For the target fish species, brook trout, bioassay mortality was most closely related to time-weighted median inorganic aluminum. Median Ca and minimum pH offered additional explanatory power, as did stream-specific aluminum responses. Due to high multicollinearity, the relative importance of different aluminum exposure duration and magnitude variables was difficult to assess, but these variables taken together added no significant explanatory power to models already containing median aluminum. Between 59 and 79% of the variation in brook trout mortality was explained by models employing between one and five regressors. Simpler models were developed for smaller sets of bioassays that tested slimy and mottled sculpin

  8. Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging. (United States)

    Li, Xueyan; Nakajima, Yoshihiro; Niwa, Kazuki; Viviani, Vadim R; Ohmiya, Yoshihiro


    A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced red-emitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37 degrees C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.

  9. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Zacharewski, T. [Univ. of Western Ontario, London, Ontario (Canada). Dept. of Pharmacology and Toxicology


    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  10. Assessing arsenic bioavailability through the use of bioassays (United States)

    Diesel, E.; Nadimpalli, M.; Hull, M.; Schreiber, M. E.; Vikesland, P.


    Various methods have been used to characterize the bioavailability of a contaminant, including chemical extractions from soils, toxicity tests, bioaccumulation measurements, estimation from soil properties, in vitro/in vivo tests, and microbial biossays. Unfortunately, these tests are all unique (i.e. they measure bioavailability through different mechanisms) and it is difficult to compare measurements collected using one method to those collected from another. Additionally, there are fundamental aspects of bioavailability research that require further study. In particular, changes in bioavailability over time are not well understood, as well as what the geochemical controls are on changes in bioavailability. In addition, there are no studies aimed at the integration of bioavailability measurements and potential geochemical controls. This research project seeks to find a standard set of assays and sensors that can be used to assess arsenic bioavailability at any field site, as well as to use these tools and techniques to better understand changes in, and controls on, arsenic bioavailability. The bioassays to be utilized in this research are a bioluminescent E. coli assay and a Corbicula fluminea (Asian clam) assay. Preliminary experiments to determine the suitability of the E. coli and C. fluminea assays have been completed. The E. coli assay can be utilized to analyze As(III) and As(V) with a linear standard curve between 5 and 200 ppb for As(III) and 100 ppb and 5 ppm for As(V); no bioluminescent response above background was elicited in the presence of Roxarsone, an organoarsenical. The C. fluminea assay is capable of bioaccumulating As(III), As(V), Roxarsone, and MSMA, with As(III) being the most readily accumulated, followed by As(V), Roxarsone and MSMA, respectively. Additional research will include assessing bioavailability of various arsenic species adsorbed to natural colloidal materials (i.e. clays, iron oxides, NOM) to the E. coli and C. fluminea assays

  11. Standardization of a fluconazole bioassay and correlation of results with those obtained by high-pressure liquid chromatography.


    Rex, J H; Hanson, L H; Amantea, M A; Stevens, D.A.; BENNETT,J.E.


    An improved bioassay for fluconazole was developed. This assay is sensitive in the clinically relevant range (2 to 40 micrograms/ml) and analyzes plasma, serum, and cerebrospinal fluid specimens; bioassay results correlate with results obtained by high-pressure liquid chromatography (HPLC). Bioassay and HPLC analyses of spiked plasma, serum, and cerebrospinal fluid samples (run as unknowns) gave good agreement with expected values. Analysis of specimens from patients gave equivalent results b...

  12. Improvement of Chemically-activated Luciferase Gene Expression Bioassay for Detection of Dioxin-like Chemicals

    Institute of Scientific and Technical Information of China (English)


    To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs. Method A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-Odeethylase (EROD) activity induction assay. Result The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.1 lpmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%,Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.

  13. Application of bioassay technique to determine onduty herbicide resistance in soil (United States)

    Bakar, F. A. A.; Ismail, B. S.; Bajrai, F. S. M.


    A study was conducted to determine the resistance of OnDuty herbicide in paddy soil with different concentrations by using a broadleaf plant, Brassica juncea. The herbicide was used in the Clearfield® Production System that was adopted in Malaysia to overcome problems mainly caused by weedy rice. Evaluation of herbicide half-life was based on bioassay technique with different concentrations, i.e 0% (control), 50% (half dose), 100% (recommended dose) and 200% (double dose). The study was done in three replicates and followed the Complete Randomized Block Design (CRBD). Results showed that there was a correlation between the amount of herbicide doses and degradation period. The highest half-life value was shown by root inhibition in the double dose concentration of 33 days half-life, followed by the recommended dose with 23 days half-life. Meanwhile, the half dose treatment indicated a half-life value of 17 days for root and 11 days for shoot. Therefore, application of herbicides should follow the recommended dose as the degradation period will not be too long, hence providing maximum effectiveness of the herbicide to overcome weed infestation problems.

  14. Predicting adverse drug reactions using publicly available PubChem BioAssay data. (United States)

    Pouliot, Y; Chiang, A P; Butte, A J


    Adverse drug reactions (ADRs) can have severe consequences, and therefore the ability to predict ADRs prior to market introduction of a drug is desirable. Computational approaches applied to preclinical data could be one way to inform drug labeling and marketing with respect to potential ADRs. Based on the premise that some of the molecular actors of ADRs involve interactions that are detectable in large, and increasingly public, compound screening campaigns, we generated logistic regression models that correlate postmarketing ADRs with screening data from the PubChem BioAssay database. These models analyze ADRs at the level of organ systems, using the system organ classes (SOCs). Of the 19 SOCs under consideration, nine were found to be significantly correlated with preclinical screening data. With regard to six of the eight established drugs for which we could retropredict SOC-specific ADRs, prior knowledge was found that supports these predictions. We conclude this paper by predicting that SOC-specific ADRs will be associated with three unapproved or recently introduced drugs.

  15. Comparison of five in vitro bioassays to measure estrogenic activity in environmental waters. (United States)

    Leusch, Frederic D L; de Jager, Christiaan; Levi, Yves; Lim, Richard; Puijker, Leo; Sacher, Frank; Tremblay, Louis A; Wilson, Vickie S; Chapman, Heather F


    Bioassays are well established in the pharmaceutical industry and single compound analysis, but there is still uncertainty about their usefulness in environmental monitoring. We compared the responses of five bioassays designed to measure estrogenic activity (the yeast estrogen screen, ER-CALUX, MELN, T47D-KBluc, and E-SCREEN assays) and chemical analysis on extracts from four different water sources (groundwater, raw sewage, treated sewage, and river water). All five bioassays displayed similar trends and there was good agreement with analytical chemistry results. The data from the ER-CALUX and E-SCREEN bioassays were robust and predictable, and well-correlated with predictions from chemical analysis. The T47D-KBluc appeared likewise promising, but with a more limited sample size it was less compelling. The YES assay was less sensitive than the other assays by an order of magnitude, which resulted in a larger number of nondetects. The MELN assay was less predictable, although the possibility that this was due to laboratory-specific difficulties cannot be discounted. With standardized bioassay data analysis and consistency of operating protocols, bioanalytical tools are a promising advance in the development of a tiered approach to environmental water quality monitoring.

  16. Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations

    Institute of Scientific and Technical Information of China (English)

    Nishant A. Dafale; Uttam P. Semwal; Piyush K. Agarwal; Pradeep Sharma; G.N. Singh


    The aim of this study was to develop and validate a simple, sensitive, precise and cost-effective one-level agar diffusion (5þ1) bioassay for estimation of potency and bioactivity of Levofloxacin in pharmaceutical preparation which has not yet been reported in any pharmacopoeia. Among 16 microbial strains, Bacillus pumilus ATCC-14884 was selected as the most significant strain against Levofloxacin. Bioassay was optimized by investigating several factors such as buffer pH, inoculums concentration and reference standard concentration. Identification of Levofloxacin in commercial sample Levoflox tablet was done by FTIR spectroscopy. Mean potency recovery value for Levofloxacin in Levoflox tablet was estimated as 100.90%. A validated bioassay method showed linearity (r2 ¼ 0.988), precision (Interday RSD ¼ 1.05%, between analyst RSD ¼ 1.02%) and accuracy (101.23%, RSD ¼ 0.72%). Bioassay was correlated with HPLC using same sample and estimated potencies were 100.90%and 99.37%, respectively. Results show that bioassay is a suitable method for estimation of potency and bioactivity of Levofloxacin pharmaceutical preparations.

  17. The usefulness of a sediment bioassay with the gastropod Nassarius reticulatus in tributyltin monitoring programs. (United States)

    Laranjeiro, Filipe; Pérez, Sara; Navarro, Patricia; Carrero, José Antonio; Beiras, Ricardo


    Despite the use of tributyltin (TBT) had been banned worldwide in 2008 there is still evidence of its deleterious presence in environment. We evaluate the usefulness of a 28days sediment bioassay with Nassarius reticulatus females to monitor TBT pollution, using imposex as endpoint. In addition, butyltins were determined in sediments and tissues, and, whenever posible, imposex was assessed in native N. reticulatus at the same sites where sediments were sampled. In the bioassay, a significant increase in imposex parameters was obtained with three sediments (Vi2, Vi3, and Vi4). No correlation was found between this and TBT concentrations in sediment although good correlations were obtained for TBT in tissues, putting in evidence TBT bioavailability in sediment. A significant decrease in imposex from 2008 to 2013 in native snails was only observed at sites that did not cause any effect in the bioassay. In contrast, imposex levels in 2013 were kept as high as 2008 in one of the sites where a significant imposex increase in the bioassay was observed. The bioassay proves thus to be a practical and ecological relevant tool, as: (i) it can be conducted in sites with no native populations of snails, (ii) it provides early identification of polluted sites, anticipating future imposex levels or early identification of recovering, and (iii) it yields information on the bioavailable fraction of the TBT in the sediment. Therefore, this tool can be of extreme usefulness under the scope of recent European legislative frameworks.

  18. Characterization of chemical waste site contamination and its extent using bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, J.M.; Callahan, C.A.; Cline, J.F.; Greene, J.C.; McShane, M.C.; Miller, W.E.; Peterson, S.A.; Simpson, J.C.; Skalski, J.R.


    Bioassays were used in a three-phase research project to assess the comparative sensitivity of test organisms to known chemicals, determine if the chemical components in field soil and water samples containing unknown contaminants could be inferred from our laboratory studies using known chemicals, and to investigate kriging (a relatively new statistical mapping technique) and bioassays as methods to define the areal extent of chemical contamination. The algal assay generally was most sensitive to samples of pure chemicals, soil elutriates and water from eight sites with known chemical contamination. Bioassays of nine samples of unknown chemical composition from the Rocky Mountain Arsenal (RMA) site showed that a lettuce seed soil contact phytoassay was most sensitive. In general, our bioassays can be used to broadly identify toxic components of contaminated soil. Nearly pure compounds of insecticides and herbicides were less toxic in the sensitive bioassays than were the counterpart commercial formulations. This finding indicates that chemical analysis alone may fail to correctly rate the severity of environmental toxicity. Finally, we used the lettuce seed phytoassay and kriging techniques in a field study at RMA to demonstrate the feasibility of mapping contamination to aid in cleanup decisions. 25 references, 9 figures, 9 tables.

  19. Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Lors, Christine [Universite Lille Nord de France, 1bis rue Georges Lefevre, 59044 Lille Cedex (France); Ecole des Mines de Douai, MPE-GCE, 941 rue Charles-Bourseul, 59500 Douai (France); Centre National de Recherche sur les Sites et Sols Pollues, 930 Boulevard Lahure, BP 537, 59505 Douai Cedex (France); Ponge, Jean-Francois, E-mail: ponge@mnhn.f [Museum National d' Histoire Naturelle, CNRS UMR 7179, 4 Avenue du Petit-Chateau, 91800 Brunoy (France); Martinez Aldaya, Maite [Museum National d' Histoire Naturelle, CNRS UMR 7179, 4 Avenue du Petit-Chateau, 91800 Brunoy (France); Damidot, Denis [Universite Lille Nord de France, 1bis rue Georges Lefevre, 59044 Lille Cedex (France); Ecole des Mines de Douai, MPE-GCE, 941 rue Charles-Bourseul, 59500 Douai (France)


    Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an 'ecoscore', a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected. - The avoidance test using the soil springtail Folsomia candida is globally more sensitive to PAH contamination than acute and chronic toxicity bioassays using plants and animals but a battery of tests could reveal better in detail.

  20. Label-free nanopore proximity bioassay for platelet-derived growth factor detection. (United States)

    Zhang, Ling; Zhang, Kaixiang; Liu, Guangchao; Liu, Mengjia; Liu, Yang; Li, Jinghong


    Rapid and sensitive detection of biomarkers with ultralow concentrations remains a great challenge in disease diagnostics. Herein, we present a label-free α-hemolysin (α-HL) nanopore proximity bioassay for protein biomarker detection by a binding-induced DNA strand displacement strategy. In this bioassay, an individual target protein, platelet-derived growth factor B-chain (PDGF-BB), was selectively recognized by two oligonucleotide affinity ligands in which an output DNA was released and translocated through α-HL nanopore with a spikelike short current block. The frequency of the current block events had a linear relationship with the concentration of PDGF-BB with a wide linear dynamic range of 5 orders of magnitude and a detection limit at 500 fM. The selectivity and anti-interference capability of this bioassay show great potential for biomarker detection in bioanalytical chemistry.

  1. Toxicity Assessment of Sediments with Natural Anomalous Concentrations in Heavy Metals by the Use of Bioassay

    Directory of Open Access Journals (Sweden)

    Francisco Martín


    Full Text Available The potential toxicity in riverbed sediments was assessed with a bioassay using the bioluminescent bacteria Vibrio fischeri. The selected area was characterized by the presence of ultramafic rocks (peridotites, and the sediments had high values in Ni, Cr, and Co. For the toxicity bioassay with Vibrio fischeri, water-soluble forms were used. The results indicated that most of the samples had a very low degree of toxicity, with 10% of reduction in luminescence in relation to the control; meanwhile 25% of the samples had a moderate degree of toxicity with a reduction in luminescence between 13 and 21% in relation to the control. The toxicity index correlated significantly with the concentrations of Ni and Cr in the water extracts. This toxicity bioassay was proved to be a sensitive and useful tool to detect potential toxicity in solutions, even with anomalous concentrations in heavy metals of natural origin.

  2. Bioassay for estimating the biogenic methane-generating potential of coal samples (United States)

    Jones, E.J.P.; Voytek, M.A.; Warwick, P.D.; Corum, M.D.; Cohn, A.; Bunnell, J.E.; Clark, A.C.; Orem, W.H.


    Generation of secondary biogenic methane in coal beds is likely controlled by a combination of factors such as the bioavailability of coal carbon, the presence of a microbial community to convert coal carbon to methane, and an environment supporting microbial growth and methanogenesis. A set of treatments and controls was developed to bioassay the bioavailability of coal for conversion to methane under defined laboratory conditions. Treatments included adding a well-characterized consortium of bacteria and methanogens (enriched from modern wetland sediments) and providing conditions to support endemic microbial activity. The contribution of desorbed methane in the bioassays was determined in treatments with bromoethane sulfonic acid, an inhibitor of microbial methanogenesis. The bioassay compared 16 subbituminous coal samples collected from beds in Texas (TX), Wyoming (WY), and Alaska (AK), and two bituminous coal samples from Pennsylvania (PA). New biogenic methane was observed in several samples of subbituminous coal with the microbial consortium added, but endemic activity was less commonly observed. The highest methane generation [80????mol methane/g coal (56??scf/ton or 1.75??cm3/g)] was from a south TX coal sample that was collected from a non-gas-producing well. Subbituminous coals from the Powder River Basin, WY and North Slope Borough, AK contained more sorbed (original) methane than the TX coal sample and generated 0-23????mol/g (up to 16??scf/ton or 0.5??cm3/g) new biogenic methane in the bioassay. Standard indicators of thermal maturity such as burial depth, nitrogen content, and calorific value did not explain differences in biogenic methane among subbituminous coal samples. No original methane was observed in two bituminous samples from PA, nor was any new methane generated in bioassays of these samples. The bioassay offers a new tool for assessing the potential of coal for biogenic methane generation, and provides a platform for studying the

  3. Review of Bioassays for Monitoring Fate and Transport ofEstrogenic Endocrine Disrupting Compounds in Water

    Energy Technology Data Exchange (ETDEWEB)


    Endocrine disrupting compounds (EDCs) are recognizedcontaminants threatening water quality. Despite efforts in sourceidentification, few strategies exist for characterization or treatment ofthis environmental pollution. Given that there are numerous EDCs that cannegatively affect humans and wildlife, general screening techniques likebioassays and biosensors provide an essential rapid and intensiveanalysis capacity. Commonly applied bioassays include the ELISA and YESassays, but promising technologies include ER-CALUXa, ELRA, Endotecta,RIANA, and IR-bioamplification. Two biosensors, Endotecta and RIANA, arefield portable using non-cellular biological detection strategies.Environmental management of EDCs in water requires integration ofbiosensors and bioassays for monitoring and assessment.

  4. Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

    DEFF Research Database (Denmark)

    Blossom, Hannah Eva; Andersen, Nikolaj Gedsted; Rasmussen, Silas Anselm;


    Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity...... the stability of the intracellular toxins when kept as a cell pellet at −20°C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge...

  5. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems. (United States)

    Grossberg, S E; Kawade, Y; Kohase, M; Yokoyama, H; Finter, N


    The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of

  6. How to translate a bioassay into a screening assay for natural products: general considerations and implementation of antimicrobial screens. (United States)

    Fallarero, Adyary; Hanski, Leena; Vuorela, Pia


    Natural product sources have been a valuable provider of molecular diversity in many drug discovery programs and several therapeutically important drugs have been isolated from these. However, the screening of such materials can be very complicated due to the fact that they contain a complex mixture of secondary metabolites, but also the purified natural compounds exert a challenge for bioactivity screening. Success in identifying new therapeutics using in vitro bioassays is largely dependent upon the proper design, validation, and implementation of the screening assay. In this review, we discuss some aspects which are of significant concern when screening natural products in a microtiter plate-based format, being partly applicable to other assay formats as well, such as validation parameters, layouts for assay protocols, and common interferences caused by natural products samples, as well as various troubleshooting strategies. Examples from the field of natural product drug discovery of antibacterial compounds are discussed, and contributions from the realm of academic screenings are highlighted.

  7. Bioassay responses and effects on benthos after pilot remediations in the delta of the rivers Rhine and Meuse. (United States)

    den Besten, Pieter J; van den Brink, Paul J


    Chemical and biological monitoring was carried out for 5 years following pilot remediations at two locations in the Rhine-Meuse delta. The remediations consisted of partial excavation of the contaminated sediments, followed by applying a clean layer of sandy material on top. After the remediation, a new silty sediment top layer was formed exhibiting a lower toxicity in five sediment/sediment pore water bioassays. Compared to the unremediated sites, lower metal and PAH concentrations were found at the remediated sites, but in one location at the same time elevated HCH, PCB and HCB levels were recorded. One year after the remediation, the differences became smaller, although effects-based classification showed that the remediated site showed a higher quality up to the last year. In both remediated sites a rapid recolonization of nematodes, oligochaetes and chironomids was observed, while the recolonization by bivalves was slower. A few years after the remediation the differences decrease.

  8. Determination of Biochemical Oxygen Demand of Area Waters: A Bioassay Procedure for Environmental Monitoring (United States)

    Riehl, Matthew


    A graphical method for determining the 5-day biochemical oxygen demand (BOD5) for a body of water is described. In this bioassay, students collect a sample of water from a designated site, transport it to the laboratory, and evaluate the amount of oxygen consumed by naturally occurring bacteria during a 5-day incubation period. An accuracy check,…

  9. Development of a bioassay system for human growth hormone determination with close correlation to immunoassay. (United States)

    Maimaiti, M; Tanahashi, Y; Mohri, Z; Fujieda, K


    Serum growth hormone (GH) level is measured largely through immunoassays in clinical practice. However, a few cases with bioinactive and immunoreactive GH have also been reported. We describe here a new bioassay system for GH determination using the BaF/GM cell line, which proliferates in a dose-dependent manner on hGH addition; cell proliferation was blocked by anti-hGH antibody. This bioassay had the lowest detection limit (∼0.02 ng/ml) reported thus far and the highest specificity for GH. The bioassay results were compared with those of an immunoradiometric assay across 163 patient samples in various endocrine states. A close correlation (the ratio of bioactivity/immunoreactivity was 1.04 ± 0.33, mean ± SD) was observed between bioactivity and immunoreactivity in these samples. The newly developed system is a specific, sensitive, easy, and fast bioassay system for GH determination; we consider it useful for evaluating GH bioactivity in various endocrine states.

  10. Bayesian Analysis for Linearized Multi-Stage Models in Quantal Bioassay. (United States)

    Kuo, Lynn; Cohen, Michael P.

    Bayesian methods for estimating dose response curves in quantal bioassay are studied. A linearized multi-stage model is assumed for the shape of the curves. A Gibbs sampling approach with data augmentation is employed to compute the Bayes estimates. In addition, estimation of the "relative additional risk" and the "risk specific…

  11. Efficiency of several cultural methods and a chick bioassay to recover dry stressed Campylobacter (United States)

    The aims of the study were to evaluate the efficacy of 5 enrichment procedures for recovery of dry-atmospheric-temperature stressed C. jejuni and C. coli and determine the viable status of the non-culturable strains using a chick bioassay. Sterile chick paper pads (PP) and filter papers (FP) were i...

  12. Analytical performance and clinical utility of a bioassay for thyroid-stimulating immunoglobulins. (United States)

    Leschik, Johannes J; Diana, Tanja; Olivo, Paul D; König, Jochem; Krahn, Ulrike; Li, Yunsheng; Kanitz, Michael; Kahaly, George J


    The analytical performance and the clinical utility of a thyrotropin receptor (TSHR)-stimulating immunoglobulin (TSI) bioassay were compared with those of a TSHR-binding inhibitory immunoglobulin (TBII) assay. Limits of detection (LoD) and quantitation (LoQ), assay cutoff, and the half-maximal effective concentration (EC(50)) were measured. Dilution analysis was performed in sera of hyperthyroid patients with Graves disease (GD) during antithyroid treatment (ATD). Titer was defined as the first dilution step at which measurement of TSI or TBII fell below the assay cutoff. The LoD, LoQ, cutoff, and EC(50) of the bioassay were 251-, 298-, 814-, and 827-fold lower than for the TBII assay. There were 22%, 42%, 23%, and 14% more positive samples in the TSI bioassay at dilutions of 1:3, 1:9, 1:27, and 1:81 (P bioassay detected lower levels of TSHR autoantibodies, and the dilution analysis provided similar predictive values of both assays in GD.

  13. Applicability of a yeast bioassay in the detection of steroid esters in hair

    NARCIS (Netherlands)

    Becue, I.; Bovee, T.F.H.; Poucke, C.; Groot, M.J.; Nielen, M.W.F.; Peteghem, van C.


    The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously

  14. Development of bioassay techniques with extracts from semi-permeable membrane devices (SPMDs)

    Energy Technology Data Exchange (ETDEWEB)

    Metcalfe, T.L.; White, P.; Mackay, D.; Metcalfe, C. [Trent Univ., Peterborough, Ontario (Canada). Environmental and Resource Studies Program


    Semi-permeable membrane devices (SPMDs), consisting of polyethylene bags filled with triolein, have been used to monitor for lipophilic organic contaminants in water. Although extracts from SPMDs have most often been analyzed for concentrations of organic contaminants, there is also the potential to monitor the toxicity of these extracts using in vitro and in vivo bioassays. SPMDs were deployed for four weeks at several sites along a corridor extending from Peche Island in the Detroit River to Pelee Island in western Lake Erie to monitor the distribution of toxic organic contaminants in the water. Analysis of the extracts from the SPMDs for concentrations of PCBs and other organochlorine compounds, and polynuclear aromatic hydrocarbons (PAHs) indicated that the regions in the Detroit River within the Trenton Channel and near Zug Island were the most highly contaminated. Bioassays conducted with extracts from the SPMDs included the in vitro SOS Chromotest for genotoxic activity, an acute lethality test with Daphnia magna, and a fish embryotoxicity test with embryos of Japanese medaka (Oryzias latipes). These bioassay data generally indicated that the toxicity and concentrations of organic contaminants in the SPMD extracts were correlated. This study indicates that there is potential to use short-term bioassays of extracts from SPMDs to monitor for in situ contamination in the aquatic environment.

  15. Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

    Directory of Open Access Journals (Sweden)

    Alexander D Crawford

    Full Text Available Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS. Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

  16. Kinetic microplate bioassays for relative potency of antibiotics improved by partial Least Square (PLS) regression. (United States)

    Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Almeida, Túlia de Souza Botelho; Lourenço, Felipe Rebello


    Microbiological assays are widely used to estimate the relative potencies of antibiotics in order to guarantee the efficacy, safety, and quality of drug products. Despite of the advantages of turbidimetric bioassays when compared to other methods, it has limitations concerning the linearity and range of the dose-response curve determination. Here, we proposed to use partial least squares (PLS) regression to solve these limitations and to improve the prediction of relative potencies of antibiotics. Kinetic-reading microplate turbidimetric bioassays for apramacyin and vancomycin were performed using Escherichia coli (ATCC 8739) and Bacillus subtilis (ATCC 6633), respectively. Microbial growths were measured as absorbance up to 180 and 300min for apramycin and vancomycin turbidimetric bioassays, respectively. Conventional dose-response curves (absorbances or area under the microbial growth curve vs. log of antibiotic concentration) showed significant regression, however there were significant deviation of linearity. Thus, they could not be used for relative potency estimations. PLS regression allowed us to construct a predictive model for estimating the relative potencies of apramycin and vancomycin without over-fitting and it improved the linear range of turbidimetric bioassay. In addition, PLS regression provided predictions of relative potencies equivalent to those obtained from agar diffusion official methods. Therefore, we conclude that PLS regression may be used to estimate the relative potencies of antibiotics with significant advantages when compared to conventional dose-response curve determination.

  17. Biological screening of selected Pacific Northwest forest plants using the brine shrimp (Artemia salina) toxicity bioassay. (United States)

    Karchesy, Yvette M; Kelsey, Rick G; Constantine, George; Karchesy, Joseph J


    The brine shrimp (Artemia salina) bioassay was used to screen 211 methanol extracts from 128 species of Pacific Northwest plants in search of general cytotoxic activity. Strong toxicity (LC50  1000 µg/ml). Our subsequent studies of conifer heartwoods with strong activity confirm the assay's value for identifying new investigational leads for materials with insecticidal and fungicidal activity.

  18. The Intersection of CMOS Microsystems and Upconversion Nanoparticles for Luminescence Bioimaging and Bioassays

    Directory of Open Access Journals (Sweden)

    Liping Wei


    Full Text Available Organic fluorophores and quantum dots are ubiquitous as contrast agents for bio-imaging and as labels in bioassays to enable the detection of biological targets and processes. Upconversion nanoparticles (UCNPs offer a different set of opportunities as labels in bioassays and for bioimaging. UCNPs are excited at near-infrared (NIR wavelengths where biological molecules are optically transparent, and their luminesce in the visible and ultraviolet (UV wavelength range is suitable for detection using complementary metal-oxide-semiconductor (CMOS technology. These nanoparticles provide multiple sharp emission bands, long lifetimes, tunable emission, high photostability, and low cytotoxicity, which render them particularly useful for bio-imaging applications and multiplexed bioassays. This paper surveys several key concepts surrounding upconversion nanoparticles and the systems that detect and process the corresponding luminescence signals. The principle of photon upconversion, tuning of emission wavelengths, UCNP bioassays, and UCNP time-resolved techniques are described. Electronic readout systems for signal detection and processing suitable for UCNP luminescence using CMOS technology are discussed. This includes recent progress in miniaturized detectors, integrated spectral sensing, and high-precision time-domain circuits. Emphasis is placed on the physical attributes of UCNPs that map strongly to the technical features that CMOS devices excel in delivering, exploring the interoperability between the two technologies.

  19. Exploring the distance between nitrogen and phosphorus limitation in mesotrophic surface waters using a sensitive bioassay (United States)

    Hrustić, Enis; Lignell, Risto; Riebesell, Ulf; Frede Thingstad, Tron


    The balance in microbial net consumption of nitrogen and phosphorus was investigated in samples collected in two mesotrophic coastal environments: the Baltic Sea (Tvärminne field station) and the North Sea (Espegrend field station). For this, we have refined a bioassay based on the response in alkaline phosphatase activity (APA) over a matrix of combinations in nitrogen and phosphorus additions. This assay not only provides information on which element (N or P) is the primary limiting nutrient, but also gives a quantitative estimate for the excess of the secondary limiting element (P+ or N+, respectively), as well as the ratio of balanced net consumption of added N and P over short timescales (days). As expected for a Baltic Sea late spring-early summer situation, the Tvärminne assays (n = 5) indicated N limitation with an average P+ = 0.30 ± 0.10 µM-P, when incubated for 4 days. For short incubations (1-2 days), the Espegrend assays indicated P limitation, but the shape of the response surface changed with incubation time, resulting in a drift in parameter estimates toward N limitation. Extrapolating back to zero incubation time gave P limitation with N+ ≈ 0.9 µM-N. The N : P ratio (molar) of nutrient net consumption varied considerably between investigated locations: from 2.3 ± 0.4 in the Tvärminne samples to 13 ± 5 and 32 ± 3 in two samples from Espegrend. Our assays included samples from mesocosm acidification experiments, but statistically significant effects of ocean acidification were not found by this method.

  20. Genotoxicity of SPL (spent pot lining) as measured by Tradescantia bioassays. (United States)

    Andrade-Vieira, L F; Davide, L C; Gedraite, L S; Campos, J M S; Azevedo, H


    Spent Pot Liner (SPL) is a solid waste product generated in the process of aluminum production. Tradescantia micronuclei (Trad-MN) and stamen hair mutation (Trad-SHM) bioassays are very useful tests to assess genotoxicity of environmental pollutants. In the present study, we intended to investigate the genotoxicity of this waste with Tradescantia bioassays using leachates of SPL simulating the natural leachability of SPL in soil. The formation of micronuclei (MN) was found to be concentration dependent. MN frequency enhanced significantly with SPL treatment. In addition, SPL also appeared to increase the percentage of dyads and triads. Trad-SHM assay showed that SPL increases pink mutation events as SPL concentration increases. These results demonstrated that SPL is a cytogenotoxic agent that affects different genetic end-points (induction of micronuclei and point mutations) even at low concentration (2% and 3%).

  1. Comparison of cancer risks projected from animal bioassays to epidemiologic studies of acrylonitrile-exposed workers. (United States)

    Ward, C E; Starr, T B


    Bioassay findings have demonstrated that acrylonitrile (ACN) is a rodent carcinogen, but the available epidemiologic evidence provides little support for the human carcinogenicity of ACN. This discordance between laboratory animal and human study findings is explored by determining post hoc the statistical power of 11 epidemiologic studies of ACN-exposed workers to detect the all-site and brain cancer excesses that are projected from rodent drinking water bioassay data. At reasonable estimates of the level and duration of exposures among the occupational cohorts, a majority of the human studies had sufficient power (> 80%) to detect the projected excesses, yet such responses were consistently absent. We conclude, subject to certain caveats, that the upper bound estimate of ACN's inhalation cancer potency of 1.5 x 10(-4) per ppm is too high to be consistent with the human ACN experience.

  2. Brine shrimp bioassay: importance of correct taxonomic identification of Artemia (Anostraca) species. (United States)

    Ruebhart, David R; Cock, Ian E; Shaw, Glen R


    Despite the common use of the brine shrimp bioassay in toxicology, there is confusion in the literature regarding citation of the correct taxonomic identity of the Artemia species used. The genus Artemia, once thought to be represented by a single species Artemia salina, is now known to be composed of several bisexual species as well as parthenogenetic populations. Artemia franciscana is the best studied of the Artemia species and is considered to represent the vast majority of studies in which Artemia is used as an experimental test organism. We found that in studies referring to the use of A. salina, the zoogeography of the cyst harvest site indicated that the species used was actually A. franciscana. Those performing bioassays with Artemia need to exercise diligence in assigning correct species identification, as the identity of the test organism is an important parameter in assuring the validity of the results of the assay.

  3. Regional differences in stratum corneum reactivity to surfactants. Quantitative assessment using the corneosurfametry bioassay. (United States)

    Henry, F; Goffin, V; Maibach, H I; Piérard, G E


    The skin does not react similarly to the presence of xenobiotics over all anatomic sites. Distinct regional differences have been described for irritancy and percutaneous absorption. The present study assesses the regional variation of stratum corneum reactivity to surfactants using the corneosurfametry bioassay. Stratum corneum was harvested from 6 body sites in 20 young adults. Corneosurfametry was performed using water, 1% SLS and a 5% soap solution. Data show that the best variable to assess regional variability in irritancy is the overall difference in corneosurfametry (ODC), comparing the effect of a given surfactant with water. The dorsal hand and volar forearm were the least reactive, the neck, forehead, back and dorsal foot the most reactive, sites. It is concluded that the corneosurfametry bioassay, through the ODC variable, is a practically noninvasive tool for the evaluation of regional variation in irritancy at the level of the stratum corneum.

  4. Bioassays Against Pinewood Nematode: Assessment of a Suitable Dilution Agent and Screening for Bioactive Essential Oils


    Ana Cristina Figueiredo; Luis G. Pedro; Barroso, José G.; Maria Teresa Tinoco; Luís Silva Dias; Mendes, Marta D.; Jorge M. S. Faria; Pedro Barbosa; Manuel Mota


    Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality...

  5. Chonemorphine, stigmasterol, and ecdysterone: steroids isolated through bioassay-directed plant screening programs. (United States)

    Shah, V C; D'Sa, A S; de Souza, N J


    In a bioassay-directed screening programme of plants for the identification of active constituents the following steroids were isolated. Chonemorphine was identified as the antiamoebic and antitrichomonad principle of Chonemorpha fragrans. Stigmasterol from Coleus forskohlii and ecdysterone from Diploclisia glaucescens were inactive constituents isolated during the process of purifying the active principles of the plants. D. glaucescens roots are a high-yielding source (0.5% of the dry root weight) of ecdysterone.

  6. Pathogenic fungi and Bio-control agents: Competitive bio-assay research



    Fungi of the genus Trichoderma have a track record of being antagonist to quite of a number of agricultural important pathogens. Trichoderma have some unique characteristics that make it scientifically proven and suitable bio-control agents against varieties of pathogenic organism infecting economic food crops. Trichoderma has the advantage of being environment friendly and not hazardous to the health of human beings, livestock, soil and environment. Competitive bio-assay experiment was carri...

  7. A Rapid and Inexpensive Bioassay to Evaluate the Decontamination of Organophosphates (United States)


    anydrolase (a hydrolyzing enzyme ), and decon- taminating foam with hydrogen peroxide. Much of the research required to quantify CWA decontamination re...household cleaners as potential decontaminating agents, but only 5% bleach was effective at improving survival of insects on steel plates treated...required to obtain nearly complete decontamination of malathion. The bioassay was also used to screen common household cleaners as potential decontaminating

  8. Carcinogenic risk of copper gluconate evaluated by a rat medium-term liver carcinogenicity bioassay protocol

    Energy Technology Data Exchange (ETDEWEB)

    Abe, Masayoshi; Usuda, Koji; Hayashi, Seigo; Ogawa, Izumi; Furukawa, Satoshi [Nissan Chemical Industries Limited, Toxicology and Environmental Science Department, Biological Research Laboratories, Saitama (Japan); Igarashi, Maki [Tokyo University of Agriculture, Laboratory of Protection of Body Function, Department of Food and Nutritional Science, Graduate School of Agriculture, Tokyo (Japan); Nakae, Dai [Tokyo University of Agriculture, Laboratory of Protection of Body Function, Department of Food and Nutritional Science, Graduate School of Agriculture, Tokyo (Japan); Tokyo Metropolitan Institute of Public Health, Tokyo (Japan)


    Carcinogenic risk and molecular mechanisms underlying the liver tumor-promoting activity of copper gluconate, an additive of functional foods, were investigated using a rat medium-term liver carcinogenicity bioassay protocol (Ito test) and a 2-week short-term administration experiment. In the medium-term liver bioassay, Fischer 344 male rats were given a single i.p. injection of N-nitrosodiethylamine at a dose of 200 mg/kg b.w. as a carcinogenic initiator. Starting 2 weeks thereafter, rats received 0, 10, 300 or 6,000 ppm of copper gluconate in diet for 6 weeks. All rats underwent 2/3 partial hepatectomy at the end of week 3, and all surviving rats were killed at the end of week 8. In the short-term experiment, rats were given 0, 10, 300 or 6,000 ppm of copper gluconate for 2 weeks. Numbers of glutathione S-transferase placental form (GST-P) positive lesions, single GST-P-positive hepatocytes and 8-oxoguanine-positive hepatocytes, and levels of cell proliferation and apoptosis in the liver were significantly increased by 6,000 ppm of copper gluconate in the medium-term liver bioassay. Furthermore, hepatic mRNA expression of genes relating to the metal metabolism, inflammation and apoptosis were elevated by 6,000 ppm of copper gluconate both in the medium-term liver bioassay and the short-term experiments. These results indicate that copper gluconate possesses carcinogenic risk toward the liver at the high dose level, and that oxidative stress and inflammatory and pro-apoptotic signaling statuses may participate in its underlying mechanisms. (orig.)

  9. Bioassay- and liquid chromatography/mass spectrometry-guided acetylcholinesterase inhibitors from Picriafel-terrae

    Directory of Open Access Journals (Sweden)

    Lu Wen


    Full Text Available Background: Picria fel-terrae is a traditional Chinese medicine. Materials and Methods: A new approach to the search for acetylcholinesterase (AChE inhibitors from Picria fel-terrae is presented. Results: Bioassay- and LC-MS-guided fractionation of the ethyl acetate extract was from traditional Chinese medicine P.fel-terrae. Following primary extraction, the ethyl acetate extracts fraction of P.fel-terrae showed strong AChE inhibitory activities. So the sample was separated using highperformance liquid chromatography (HPLC. The effluent was split towards two identical 96-well fraction collectors, and the presence of the biologically interesting portion and chromatographic fractions could be readily detected by analyzing selected ion chromatograms through an electrophoresis-electrospray ionization mass spectrometry (ESIMS system for accurate mass measurement. One 96-well plate was used for a bioassay (AChE-inhibitory assay and detected the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate was used for identification by LC-(+ ESIMS. Conclusion: As abovementioned, the AChE inhibitory constituents from P.fel-terrae by LC-bioassay-ESIMS were rapid identified. Liquid chromatography/ mass spectrometry (LC-MS screening detected the presence of six active compounds, identified as picfeltarraenin IA (1, picfeltarraenin IB (2, picfeltarraenin IV (3, picfeltarraenin X (4, picfeltarraenin XI (5, and one unknown compound. The structures were further determined by 13C NMR. The six compounds expressed stronger AChE inhibition than the known AChE inhibitorTacrine. Above all, the value of this LC-bioassay-ESIMS methodology is highlighted by the finding and structure elucidation of the active constituents from many other structural families of natural products.

  10. Recombinant Streptomyces clavuligerus strain including cas2 gene production and analysis its antibiotic overproduction by bioassay

    Directory of Open Access Journals (Sweden)

    Zohreh Hojati


    Full Text Available Background: Streptomyces clavuligerus is one of the most important strain that produce clavulanic acid that wildly used in combination of strong but sensitive to β-lactamase antibiotics in clinics. The cas2 is one of the important genes in the biosynthesis pathway of clavulanic acid. Materials and Methods: The recombinant construct pMTcas2 which contain cas2 gene is obtained from Isfahan University. Recombinant plasmid extracts from streptomyces lividans and confirm by enzyme digestion. The streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR using cas2 specific primers. Finally, bioassay method was used to survey the effect of extra copy of cas2 on clavulanic acid production. Result: Plasmid extraction was initially carried out and the structure of plasmid was confirmed by digestion. The typical white colony was seen on protoplast recovery culture containing thiostrepton antibiotic and gray spores were detected after one week. Plasmid extraction was done from transformed strain and transformation was confirmed by PCR. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Conclusion: The bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Overproduction of clavulanic acid decreases the cost of its dependent drug production.

  11. Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption. (United States)

    Houtman, Corine J; Cenijn, Peter H; Hamers, Timo; Lamoree, Marja H; Legler, Juliette; Murk, Albertinka J; Brouwer, Abraham


    In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.

  12. Bioassays as one of the Green Chemistry tools for assessing environmental quality: A review. (United States)

    Wieczerzak, M; Namieśnik, J; Kudłak, B


    For centuries, mankind has contributed to irreversible environmental changes, but due to the modern science of recent decades, scientists are able to assess the scale of this impact. The introduction of laws and standards to ensure environmental cleanliness requires comprehensive environmental monitoring, which should also meet the requirements of Green Chemistry. The broad spectrum of Green Chemistry principle applications should also include all of the techniques and methods of pollutant analysis and environmental monitoring. The classical methods of chemical analyses do not always match the twelve principles of Green Chemistry, and they are often expensive and employ toxic and environmentally unfriendly solvents in large quantities. These solvents can generate hazardous and toxic waste while consuming large volumes of resources. Therefore, there is a need to develop reliable techniques that would not only meet the requirements of Green Analytical Chemistry, but they could also complement and sometimes provide an alternative to conventional classical analytical methods. These alternatives may be found in bioassays. Commercially available certified bioassays often come in the form of ready-to-use toxkits, and they are easy to use and relatively inexpensive in comparison with certain conventional analytical methods. The aim of this study is to provide evidence that bioassays can be a complementary alternative to classical methods of analysis and can fulfil Green Analytical Chemistry criteria. The test organisms discussed in this work include single-celled organisms, such as cell lines, fungi (yeast), and bacteria, and multicellular organisms, such as invertebrate and vertebrate animals and plants.

  13. Application of the CALUX bioassay for epidemiological study. Analyses of Belgian human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Wouwe, N. van; Debacker, N.; Sasse, A. [Scientific Institute of Public Health, Brussels (BE)] (and others)


    The CALUX bioassay is a promising screening method for the detection of dioxin-like compounds. The observed good sensitivity, low number of false negative results as well as the good correlations with the GC-HRMS TEQ-values in case of feed and food analyses allow this method to climb in the first assessment methods' scale. The low amount of sample needed in addition to those latest advantages suggest that the CALUX bioassay could be a good screening method for epidemiological studies. The Belgian epidemiological study concerning the possible effect of the dioxin incident on the body burden of the Belgian population was an opportunity to test this method in comparison to the gold reference one: the GC-HRMS. The first part of this abstract presents epidemiological parameters (sensibility, specificity,) of the CALUX bioassay using CALUX TEQ-values as estimators of the TEQ-values of the 17 PCDD/Fs. The second part examines epidemiological determinants observed for CALUX and GCHRMS TEQ-values.

  14. Eliminating bias in routine bioassay when there is unknown time of intake

    Energy Technology Data Exchange (ETDEWEB)

    Strom, Daniel J.


    Routine bioassay programs sometimes find evidence of an unsuspected intake. If there were no workplace indicators of exposure or intake, it is necessary to assume a value for the time of intake. Under these circumstances, the International Commission on Radiological Protection (ICRP) continues to recommend using the midpoint of the interval between routine bioassay measurements (ICRP Pub. 78, para. 106). The assumption of T/2 as the time of intake, where T is the interval between bioassay measurements, represents the expectation value of the time of intake, , assuming uniform probability of an intake at any given time. In virtually all cases presented here, this assumption results in a significant underestimation, in many cases by a factor of 2 or 3, of the expectation value of the intake, , that would have been received by a population of workers who had uniform probability over time of intake. This underestimation leads to a negative bias in dose estimates derived in this fashion. The bias is characterized for realistic, routine urinalysis programs for Pu, U, and 3H, as well as for in vivo measurements of 125I and 137Cs. Simple methods are presented for finding t, the time of using software such as IMBA, LUDEP, or CINDY. Since the primary concern is estimating intake rather than time, the assumed time of intake should be chosen as t rather than . The ICRP should consider revising some of the tables in its Publication 78 to reflect this.

  15. Assessing the genotoxicity of urban air pollutants using two in situ plant bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Villarini, M.; Fatigoni, C.; Dominici, L.; Maestri, S. [Department of Medical-Surgical Specialties and Public Health, University of Perugia, I-06126 (Italy); Ederli, L.; Pasqualini, S. [Department of Applied Biology, University of Perugia, I-06121 (Italy); Monarca, S. [Department of Medical-Surgical Specialties and Public Health, University of Perugia, I-06126 (Italy); Moretti, M., E-mail: massimo.moretti@unipg.i [Department of Medical-Surgical Specialties and Public Health, University of Perugia, I-06126 (Italy)


    Genotoxicity of urban air has been analysed almost exclusively in airborne particulates. We monitored the genotoxic effects of airborne pollutants in the urban air of Perugia (Central Italy). Two plant bioindicators with different genetic endpoints were used: micronuclei in meiotic pollen mother cells using Tradescantia-micronucleus bioassay (Trad-MCN) and DNA damage in nuclei of Nicotiana tabacum leaves using comet assay (Nicotiana-comet). Buds of Tradescantia clone no. 4430 and young N. tabacum cv. Xanthi plants were exposed for 24 h at three sites with different pollution levels. One control site (indoor control) was also used. The two bioassays showed different sensitivities toward urban pollutants: Trad-MCN assay was the most sensitive, but DNA damage in N. tabacum showed a better correlation with the pollutant concentrations. In situ biomonitoring of airborne genotoxins using higher plants combined with chemical analysis is thus recommended for characterizing genotoxicity of urban air. - Plant bioassays used to explore in situ the correlation between air pollution and genotoxicity.

  16. Comparison of the sensitivity of seven marine and freshwater bioassays as regards antidepressant toxicity assessment. (United States)

    Minguez, Laetitia; Di Poi, Carole; Farcy, Emilie; Ballandonne, Céline; Benchouala, Amira; Bojic, Clément; Cossu-Leguille, Carole; Costil, Katherine; Serpentini, Antoine; Lebel, Jean-Marc; Halm-Lemeille, Marie-Pierre


    The hazards linked to pharmaceutical residues like antidepressants are currently a major concern of ecotoxicology because they may have adverse effects on non-target aquatic organisms. Our study assesses the ecotoxicity of three antidepressants (fluoxetine, sertraline and clomipramine) using a battery of marine and freshwater species representing different trophic levels, and compares the bioassay sensitivity levels. We selected the following bioassays: the algal growth inhibition test (Skeletonema marinoi and Pseudokirchneriella subcapitata), the microcrustacean immobilization test (Artemia salina and Daphnia magna), development and adult survival tests on Hydra attenuata, embryotoxicity and metamorphosis tests on Crassostrea gigas, and in vitro assays on primary cultures of Haliotis tuberculata hemocytes. The results showed high inter-species variability in EC50-values ranging from 43 to 15,600 µg/L for fluoxetine, from 67 to 4,400 µg/L for sertraline, and from 4.70 µg/L to more than 100,000 µg/L for clomipramine. Algae (S. marinoi and P. subcapitata) and the embryo-larval stages of the oyster C. gigas were the most sensitive taxa. This raises an issue due to their ecological and/or economic importance. The marine crustacean A. salina was the least sensitive species. This difference in sensitivity between bioassays highlights the importance of using a test battery.

  17. Glycomics Approaches for the Bioassay and Structural Analysis of Heparin/Heparan Sulphates

    Directory of Open Access Journals (Sweden)

    Jeremy E. Turnbull


    Full Text Available The glycosaminoglycan heparan sulphate (HS has a heterogeneous structure; evidence shows that specific structures may be responsible for specific functions in biological processes such as blood coagulation and regulation of growth factor signalling. This review summarises the different experimental tools and methods developed to provide more rapid methods for studying the structure and functions of HS. Rapid and sensitive methods for the facile purification of HS, from tissue and cell sources are reviewed. Data sets for the structural analysis are often complex and include multiple sample sets, therefore different software and tools have been developed for the analysis of different HS data sets. These can be readily applied to chromatographic data sets for the simplification of data (e.g., charge separation using strong anion exchange chromatography and from size separation using gel filtration techniques. Finally, following the sequencing of the human genome, research has rapidly advanced with the introduction of high throughput technologies to carry out simultaneous analyses of many samples. Microarrays to study macromolecular interactions (including glycan arrays have paved the way for bioassay technologies which utilize cell arrays to study the effects of multiple macromolecules on cells. Glycan bioassay technologies are described in which immobilisation techniques for saccharides are exploited to develop a platform to probe cell responses such as signalling pathway activation. This review aims at reviewing available techniques and tools for the purification, analysis and bioassay of HS saccharides in biological systems using “glycomics” approaches.

  18. A Chemo Attractant in Onion Root Exudates Recognized by Ditylenchus dipsaci in Laboratory Bioassay. (United States)

    Spiegel, Y; Burrows, P M; Bar-Eyal, M


    ABSTRACT A quantitative bioassay that translates preferences of axenically cultured and field population of Ditylenchus dipsaci, observed in vitro, into relative attractiveness of sterile root exudates preparations and their components is described. Onion (Allium cepa cv. White Lisbon) root exudates (ORE) are consistently and significantly much more attractive than the buffer control in all these assays. Exudates from oat cv. Lodi, mustard cv. Albatross and tomato cv. Rehovot 13 are significantly more attractive than the buffer but less attractive than ORE; Arabidopsis sp. cv. Landsberg erecta, oil seed rape cv. Cetes and wheat cv. Bet Hashita are as attractive as the buffer, but canary grass and clover exudates are less attractive than the buffer and, therefore, are classified as repellent. No significant differences in relative attractiveness were detected among exudates from other two cultivars of onion (Texas Grano 502 and Granex Hybrid) and one cultivar of leek (Large American Flag), but exudates from one onion (cv. Evergreen Long White Bunching) and one leek (cv. Broad London) were less attractive than ORE. Relative attractiveness is linear in relation to dilution exponent and therefore log-linear in relation to ORE concentration. Host (onion) penetration study reveals that penetration preferences by D. dipsaci follow the same pattern as those predicted by relative attractiveness coefficients estimated in the bio-assays. Preliminary characterization of the chemo attractant from ORE, using the behavioral bioassay, demonstrated that it was stable to heat and to proteolytic enzymes, nonvolatile and water soluble with a molecular mass <700 kDa.

  19. Noninvasive quantitation of human liver steatosis using magnetic resonance and bioassay methods

    Energy Technology Data Exchange (ETDEWEB)

    D' Assignies, Gaspard; Ruel, Martin; Khiat, Abdesslem; Lepanto, Luigi; Kauffmann, Claude; Tang, An [Centre Hospitalier de l' Universite de Montreal (CHUM), Departement de Radiologie, Montreal, Quebec (Canada); Chagnon, Miguel [Universite de Montreal (UDEM), Departement de Mathematiques et de Statistique, Montreal, Quebec (Canada); Gaboury, Louis [Centre Hospitalier de l' Universite de Montreal (CHUM), Departement d' Anatomo-Pathologie, Montreal, Quebec (Canada); Boulanger, Yvan [Centre Hospitalier de l' Universite de Montreal (CHUM), Departement de Radiologie, Montreal, Quebec (Canada); Hopital Saint-Luc du CHUM, Departement de Radiologie, Montreal, Quebec (Canada)


    The purpose was to evaluate the ability of three magnetic resonance (MR) techniques to detect liver steatosis and to determine which noninvasive technique (MR, bioassays) or combination of techniques is optimal for the quantification of hepatic fat using histopathology as a reference. Twenty patients with histopathologically proven steatosis and 24 control subjects underwent single-voxel proton MR spectroscopy (MRS; 3 voxels), dual-echo in phase/out of phase MR imaging (DEI) and diffusion-weighted MR imaging (DWI) examinations of the liver. Blood or urine bioassays were also performed for steatosis patients. Both MRS and DEI data allowed to detect steatosis with a high sensitivity (0.95 for MRS; 1 for DEI) and specificity (1 for MRS; 0.875 for DEI) but not DWI. Strong correlations were found between fat fraction (FF) measured by MRS, DEI and histopathology segmentation as well as with low density lipoprotein (LDL) and cholesterol concentrations. A Bland-Altman analysis showed a good agreement between the FF measured by MRS and DEI. Partial correlation analyses failed to improve the correlation with segmentation FF when MRS or DEI data were combined with bioassay results. Therefore, FF from MRS or DEI appear to be the best parameters to both detect steatosis and accurately quantify fat liver noninvasively. (orig.)


    NTPBSI: National Toxicology Program Bioassay On-line Database Structure-Index Locator File. Database contains the results collected on approxiately 300 toxicity studies from shorter duration test and from genetic toxicity studies, both in vitro and in vivo tests.

  1. Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty. (United States)

    Saviano, Alessandro Morais; Francisco, Fabiane Lacerda; Ostronoff, Celina Silva; Lourenço, Felipe Rebello


    The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

  2. Pharmacokinetic study with N-Ile1-Thr2-63-desulfato-r-hirudin in rabbits by means of bioassay

    Institute of Scientific and Technical Information of China (English)


    Aim: To study the pharmacokinetic (PK) properties in rabbits treated with N-Ile1-Thr2-63-desulfato-r-hirudin (rH) newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent. Methods: rH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg. Results: The bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows: t1/2β 58.4~59 min. Vd 0.09~0.12 L/kg, CL 0.0035~0.0040 L/(kg.min);AUC were proportional to the doses, t1/2 and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t1/2β and 13 -fold greater A UC than normal healthy rabbits.Conclusion: rH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site.The excretion ofrH by kidneys played a very important role in the elimination of rH. The PK ofrH could be described by the twoand one-compartment model after iv and sc dosing, respectively, and followed linear kinetics.

  3. Comparison of mathematically-predicted toxic equivalents (TEQs) and bioassay-derived dioxin-equivalents (TCDD-EQs) in heron embryos (United States)

    Rattner, B.; Hatfield, J.; Melancon, M.; Custer, T.; Tillitt, D.


    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from an uncontaminated site (Chincoteague National Wildlife Refuge,VA) and three polluted sites (Cat Island, Green Bay, WI; Bair and West Marin Islands, San Francisco Bay, CA). Hepatic microsomal monooxygenases were induced up to 85-fold relative to the reference site, and was associated with concentrations of total PCBs and 11 PCB congeners that are presumed to express toxicity through the Ah receptor. TEQs [mathematically predicted; summed product of PCB congener concentrations using 5 different sets of toxic equivalency factors (TEFs)] were compared to TCDD-EQs [derived by bioassay; ethoxyresorufin-O-dealkylase activity of treated H411E rat hepatoma cells]. Although TEQs were up to 15-fold greater than TCDD-EQs, the pattern among sites was consistent and TEQs were highly correlated with TCDD-EQs. TEFs based on single congener mammalian studies yielded TEQs that greatly exceeded values from the H411E bioassay of field sample. TEFs generated from avian egg injection studies yielded TEQs that most closely approximated bioassay-derived TCDD-EQs. Cytochrome P450 parameters were related to TEQs and TCDD-EQs; adjusted r2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data document the general predictive value of TEQs and TCDD-EQs for P450 induction in field collected samples, but also indicate the need for development of TEFs for the species and biological end point of concern.

  4. Laboratory Bioassays with Three Different Substrates to Test the Efficacy of Insecticides against Various Stages of Drosophila suzukii (Diptera: Drosophilidae) (United States)

    Pavlova, Aneliya Koleva; Dahlmann, Melanie; Hauck, Mirjam


    Rapid worldwide spread and polyphagous nature of the spotted wing Drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae) calls for efficient and selective control strategies to prevent severe economic losses in various fruit crops. The use of insecticides is one option for management of this invasive pest insect. Efficacy of insecticides is usually assessed first in laboratory bioassays, which are compounded by the cryptic nature of D. suzukii larvae and the fact that fruits used in bioassays often start to rot and dissolve before larvae have reached the adult stage. Here, we report on laboratory bioassays using three different types of substrates allowing a thorough screening of insecticides for their potential effects against D. suzukii eggs, larvae and adults. Suitability of our bioassays was validated in an assessment of the efficacy of four bioinsecticides and one synthetic insecticide against various developmental stages of D. suzukii. Water-apple juice agar used as a bioassay substrate allowed egg counting and observation of larval development due to its transparency, while apple-nutrition medium allowed complete metamorphosis. Use of grape berries in bioassays made it possible to assess effects of an insecticide present on a fruit’s surface on oviposition and larval hatch from eggs. Insecticides tested in these three different bioassays with acetamiprid, spinosad or natural pyrethrins as active ingredients achieved a significant D. suzukii control if they were applied before egg deposition. Number of adult flies was significantly reduced if the bioassay medium was treated with an azadirachtin A containing insecticide both before or after egg deposition. PMID:28042104

  5. The H4IIE cell bioassay as an indicator of dioxin-like chemicals in wildlife and the environment. (United States)

    Whyte, J J; Schmitt, C J; Tillitt, D E


    The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

  6. Laboratory bioassay for assessing the effects of sludge supernatant on plant growth and vesicular-arbuscular mycorrhiza formation

    Energy Technology Data Exchange (ETDEWEB)

    Bohn, K.S.; Liberta, A.E.


    A laboratory bioassay is described for assessing the effects of sludge supernatant on juvenile corn growth and the ability of vesicular-arbuscular (VA) mycorrhizal fungi, indigenous to coal spoil, to form mycorrhizae. The bioassay demonstrated that application rates can be identified that have the potential to promote increased plant dry weight without suppressing the formation of VA mycorrhizae in a plant's root system.

  7. Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"


    Ambrosiadis Ioannis; Dasiou Despina; Filioussis George; Avramidis Nicholaos; Papagianni Maria


    Abstract Background Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried o...

  8. Liposomes as signal amplification reagents for bioassays in microfluidic channels. (United States)

    Locascio, Laurie E; Hong, Jennifer S; Gaitan, Michael


    Liposomes with encapsulated carboxyfluorescein were used in an affinity-based assay to provide signal amplification for small-volume fluorescence measurements. Microfluidic channels were fabricated by imprinting in a plastic substrate material, poly(ethylene terephthalate glycol) (PETG), using a silicon template imprinting tool. Streptavidin was linked to the surface through biotinylated-protein for effective immobilization with minimal nonspecific adsorption of the liposome reagent. Lipids derivatized with biotin were incorporated into the liposome membrane to make the liposomes reactive for affinity assays. Specific binding of the liposomes to microchannel walls, dependence of binding on incubation time, and nonspecific adsorption of the liposome reagent were evaluated. The results of a competitive assay employing liposomes in the microchannels are presented.

  9. A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity. (United States)

    Ramanathan, Palaniappan; Zhu, James J; Bishop, Elizabeth A; Puckette, Michael C; Hartwig, Ethan; Grubman, Marvin J; Rodriguez, Luis L


    Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

  10. A rapid and high-throughput quantum dots bioassay for monitoring of perfluorooctane sulfonate in environmental water samples

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Jiong; Wan Yanjian; Li Yuanyuan; Zhang Qiongfang; Xu Shunqing [Minister of Education Key Laboratory of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030 (China); Zhu Huijun [Cranfield Health, Cranfield University, Kempston, Bedfordshire, MK43 0AL (United Kingdom); Shu Baihua, E-mail: [Minister of Education Key Laboratory of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030 (China)


    Currently HPLC/MS is the state of the art tool for environmental/drinking water perfluorooctane sulfonate (PFOS) monitoring. PFOS can bind to peroxisomal proliferator-activated receptor-alpha (PPAR{alpha}), which forms heterodimers with retinoid X receptors (RXRs) and binds to PPAR response elements. In this bioassay free PFOS in water samples competes with immobilized PFOS in ELISA plates for a given amount of PPAR{alpha}-RXR{alpha}. It can be determined indirectly by immobilizing PPAR{alpha}-RXR{alpha}-PFOS complex to another plate coated with PPAR{alpha} antibody and subsequent measuring the level of PPAR{alpha}-RXR{alpha} by using biotin-modified PPAR{alpha}-RXR{alpha} probes-quantum dots-streptavidin detection system. The rapid and high-throughput bioassay demonstrated a detection limit of 2.5 ng L{sup -1} with linear range between 2.5 ng L{sup -1} and 75 ng L{sup -1}. Detection results of environmental water samples were highly consistent between the bioassay and HPLC/MS. - We developed a rapid and high-throughput bioassay for monitoring of PFOS in environmental water samples. - Highlights: > We developed a rapid and high-throughput bioassay for monitoring of PFOS in water. > We detected the PFOS concentration of water samples by two methods. > The bioassay is effective for evaluating PFOS contamination level.

  11. Earthworm Preference Bioassays to Evaluate Land Management Practices. (United States)

    Bouldin, Jennifer L; Klasky, John W P; Green, V Steven


    Earthworm preference tests, especially in soil-dosed exposures, can be an informative tool for assessing land management practices. Agricultural management intended to increase crop yield and improve soil sustainability includes physical manipulation of topsoil through conventional tillage, reduced or no-tillage, and/or winter cover crops. Soil amendments include the addition of inorganic nitrogen or organic nitrogen derived from soil amendments including biosolids from sewage treatment plants, poultry litter, or locally available industrial effluent. This study used 48-h Eisenia fetida preference tests to assess impacts of agricultural management practices on soil macrofauna. Although in laboratory-dosed exposures, E. fetida preferred biosolid-dosed soils (80 %-95 % recovery) over control soils, the same results were not found with field soils receiving biosolid amendments (33 % recovery). Poultry litter-amended soils (68 % recovery) were preferred over control soils. No differences were measured between tilled fields and controls, and earthworms preferred control soils over those from fields with no-tillage and cover crops. Soil assessments through laboratory exposures such as these allows science-based agricultural management decisions to maintain or improve soil health.

  12. Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles. (United States)

    Jin, Ling; Escher, Beate I; Limpus, Colin J; Gaus, Caroline


    Conventional target analysis of biological samples such as blood limits our ability to understand mixture effects of chemicals. This study aimed to establish a rapid passive sampling technique using the polymer polydimethylsiloxane (PDMS) for exhaustive extraction of mixtures of neutral organic chemicals accumulated in blood of green turtles, in preparation for screening in in vitro bioassays. We designed a PDMS-blood partitioning system based on the partition coefficients of chemicals between PDMS and major blood components. The sampling kinetics of hydrophobic test chemicals (polychlorinated dibenzo-p-dioxins; PCDDs) from blood into PDMS were reasonably fast reaching steady state in turtles with known concentrations of PCDD/Fs, dioxin-like PCBs, PBDEs and organochlorine pesticides. The quantified chemicals explained most of the dioxin-like activity (69-98%), but less than 0.4% of the oxidative stress response. The results demonstrate the applicability of PDMS-based passive sampling to extract bioaccumulative chemicals from blood as well as the value of in vitro bioassays for capturing the combined effects of unknown and known chemicals.

  13. Assessing biochar ecotoxicology for soil amendment by root phytotoxicity bioassays. (United States)

    Visioli, Giovanna; Conti, Federica D; Menta, Cristina; Bandiera, Marianna; Malcevschi, Alessio; Jones, Davey L; Vamerali, Teofilo


    Soil amendment with biochar has been proposed as effective in improving agricultural land fertility and carbon sequestration, although the characterisation and certification of biochar quality are still crucial for widespread acceptance for agronomic purposes. We describe here the effects of four biochars (conifer and poplar wood, grape marc, wheat straw) at increasing application rates (0.5, 1, 2, 5, 10, 20, 50% w/w) on both germination and root elongation of Cucumis sativus L., Lepidium sativum L. and Sorghum saccharatum Moench. The tested biochars varied in chemical properties, depending on the type and quality of the initial feedstock batch, polycyclic aromatic hydrocarbons (PAHs) being high in conifer and wheat straw, Cd in poplar and Cu in grape marc. We demonstrate that electrical conductivity and Cu negatively affected both germination and root elongation at ≥5% rate biochar, together with Zn at ≥10% and elevated pH at ≥20%. In all species, germination was less sensitive than root elongation, strongly decreasing at very high rates of chars from grape marc (>10%) and wheat straw (>50%), whereas root length was already affected at 0.5% of conifer and poplar in cucumber and sorghum, with marked impairment in all chars at >5%. As a general interpretation, we propose here logarithmic model for robust root phytotoxicity in sorghum, based on biochar Zn content, which explains 66% of variability over the whole dosage range tested. We conclude that metal contamination is a crucial quality parameter for biochar safety, and that root elongation represents a stable test for assessing phytotoxicity at recommended in-field amendment rates (<1-2%).

  14. Bacterial and enzymatic bioassays for toxicity testing in the environment. (United States)

    Bitton, G; Koopman, B


    Microbioassays using bacteria or enzymes are increasingly applied to measure chemical toxicity in the environment. Attractive features of these assays may include low cost, rapid response to toxicants, high sample throughput, modest laboratory equipment and space requirements, low sample volume, portability, and reproducible responses. Enzymatic tests rely on measurement of either enzyme activity or enzyme biosynthesis. Dehydrogenases are the enzymes most used in toxicity testing. Assay of dehydrogenase activity is conveniently carried out using oxidoreduction dyes such as tetrazolium salts. Other enzyme activity tests utilize ATPases, esterases, phosphatases, urease, luciferase, beta-galactosidase, protease, amylase, or beta-glucosidase. Recently, the inhibition of enzyme (beta-galactosidase, tryptophanase, alpha-glucosidase) biosynthesis has been explored as a basis for toxicity testing. Enzyme biosynthesis was found to be generally more sensitive to organic chemicals than enzyme activity. Bacterial toxicity tests are based on bioluminescence, motility, growth, viability, ATP, oxygen uptake, nitrification, or heat production. An important aspect of bacterial tests is the permeability of cells to environmental toxicants, particularly organic chemicals of hydrophobic nature. Physical, chemical, and genetic alterations of the outer membrane of E. coli have been found to affect test sensitivity to organic toxicants. Several microbioassays are now commercially available. The names of the assays and their basis are: Microtox (bioluminescence), Polytox (respiration), ECHA Biocide Monitor (dehydrogenase activity), Toxi-Chromotest (enzyme biosynthesis), and MetPAD (enzyme activity). An important feature common to these tests is the provision of standardized cultures of bacteria in freeze-dried form. Two of the more recent applications of microbioassays are in sediment toxicity testing and toxicity reduction evaluation. Sediment pore water may be assayed directly or

  15. Anaerobic bioassay of methane potential of microalgal biomass (United States)

    Yen, Hong-Wei

    This study was undertaken to investigate the feasibility of using anaerobic digestion as a technique to recover solar energy embodied in excess algal biomass production harvested from Clemson University's high rate algal based Partitioned Aquaculture System (PAS) as an energy source to support PAS operations. In this study, four different organic substrates were loaded to anaerobic digesters in eight experimental trials, to ascertain the optimal combination of operational variables and effect of algal, or modified algal substrate upon methane production rate. The four substrates used in this study were: (1) a synthetic feedstock consisting of molasses and dog food, (2) a commercially obtained, readily degradable algal biomass (Spirulina ) in dry form, (3) PAS harvested and dewatered algal sludge, and (4) algal biomass blended with shredded waste paper or molasses as a carbon supplement for the adjustment of algal C/N ratio. Eight experimental trials using combinations of the four substrates were conducted in 15 liter digesters to investigate the effects of controlled digester parameters upon digester performance. Digesters operating at 20 days HRT, mesophilic digestion (35°C), and twice per day mixing at maximal loading rates produced maximal methane gas using PAS algal sludge. However, under these conditions overall methane production was less than 1000 ml CH4/l day. This low level of energy recovery from the fermentation of algal biomass (alone) is not energetically or economically favorable. Co-digestion of algal sludge and waste paper was investigated as a way to increase methane production. The data obtained from these trials suggest an optimum C/N ratio for co-digestion of algal sludge and waste paper in the range of 20--25/l. A balanced C/N ratio along with the stimulated increase in cellulase activity is suggested as likely reasons for increased methane production seen in co-digestion of algal sludge and waste paper. Yeast extract addition to anaerobic

  16. Bioassays for estrogenic activity: development and validation of estrogen receptor (ERalpha/ERbeta) and breast cancer proliferation bioassays to measure serum estrogenic activity in clinical studies. (United States)

    Li, J; Lee, L; Gong, Y; Shen, P; Wong, S P; Wise, Stephen D; Yong, E L


    Standard estrogenic prodrugs such as estradiol valerate (E2V) and increasingly popular phytoestrogen formulations are commonly prescribed to improve menopausal health. These drugs are metabolized to numerous bioactive compounds, known or unknown, which may exert combinatorial estrogenic effects in vivo. The aim of this study is to develop and validate estrogen receptor (ER) alpha/ERbeta reporter gene and MCF-7 breast cancer cell proliferation bioassays to quantify serum estrogenic activities in a clinical trial setting. We measured changes in serum estrogenicity following ingestion of E2V and compared this to mass spectrometric measurements of its bioactive metabolites, estrone and 17beta-stradiol. ERalpha bioactivity of the 192 serum samples correlated well (R = 79%) with 17beta-estradiol levels, and adding estrone improved R to 0.83 (likelihood ratio test, P estrogenic activity and that these assays suggest that the Epimedium formulation tested is unlikely to exert significant estrogenic effects in humans.

  17. Characterization and bioassay for larvicidal activity of Anacardium occidentale (cashew) shell waste fractions against dengue vector Aedes aegypti. (United States)

    Torres, Rosalinda C; Garbo, Alicia G; Walde, Rikkamae Zinca Marie L


    Recent studies regarding the harmful effects of synthetic larvicides initiated the need to investigate for unconventional measures that are environmentally safe and target-specific against Aedes aegypti larvae. Thus, the main objectives of the study are to evaluate the larvicidal toxicity of the solvent fractions of Anacardium occidentale shell wastes against the third and fourth instar larvae of A. aegypti and to compare the results with the commercial larvicide product. The shell wastes were extracted with 95% EtOH followed by polarity-based fractionation. The fractions were tested for larvicidal activity according to the World Health Organization bioassay method. These were then characterized by quantitative thin-layer chromatographic (TLC) fingerprinting. The hexane fraction gave the strongest activity among the fractions with an LC50 of 4.01 mg/L and LC90 of 11.29 mg/L highly comparable to the commercial larvicide, which exhibited an LC50 of 1.71 mg/L and LC90 of 8.41 mg/L. The dichloromethane fraction exhibited 9.70 mg/L LC50 and 18.44 mg/L LC90. The remarkable toxicity effects exhibited by these fractions indicate their potential to provide core structures from which sustainable and environmentally safe plant-based larvicidal agents can be synthesized.

  18. Oversimplification and overstandardization in biological methods: sperm bioassays in ecotoxicology as a case of study and a proposal for their reformulation. (United States)

    Murado, M A; Prieto, M A


    An interesting toxicological bioassay (fertilization inhibition in sea urchin) uses as assessment criterion a variable (fertilization ratio) whose variation with time creates two types of difficulties. First, it fails to distinguish between the toxic effect and the spontaneous decline in the sperm activity, causing some inconsistencies. Second, the sensitivity of the fertilization ratio to many other variables of the system requires a complex standardization, constraining the achievement of the method without solving its main problem. Our proposal consists of using a parameter (sperm half-life) as the response of the assay, and describing explicitly the behavior of the system as a simultaneous function of dose and time. This new focus is able to solve the problematic character of the results based on the fertilization ratio and by using the same data set which is required by the conventional approach; it simplifies the protocol, economizes experimental effort, provides unambiguous and robust results, and contributes to the detection of an artefactual temperature effect, which is not very evident under the usual perspective. Potential application of this new approach to the improvement of other formally similar bioassays is finally suggested.

  19. Oversimplification and Overstandardization in Biological Methods: Sperm Bioassays in Ecotoxicology as a Case of Study and a Proposal for Their Reformulation

    Directory of Open Access Journals (Sweden)

    M. A. Murado


    Full Text Available An interesting toxicological bioassay (fertilization inhibition in sea urchin uses as assessment criterion a variable (fertilization ratio whose variation with time creates two types of difficulties. First, it fails to distinguish between the toxic effect and the spontaneous decline in the sperm activity, causing some inconsistencies. Second, the sensitivity of the fertilization ratio to many other variables of the system requires a complex standardization, constraining the achievement of the method without solving its main problem. Our proposal consists of using a parameter (sperm half-life as the response of the assay, and describing explicitly the behavior of the system as a simultaneous function of dose and time. This new focus is able to solve the problematic character of the results based on the fertilization ratio and by using the same data set which is required by the conventional approach; it simplifies the protocol, economizes experimental effort, provides unambiguous and robust results, and contributes to the detection of an artefactual temperature effect, which is not very evident under the usual perspective. Potential application of this new approach to the improvement of other formally similar bioassays is finally suggested.

  20. Understanding bioavailability and toxicity of sediment-associated contaminants by combining passive sampling with in vitro bioassays in an urban river catchment. (United States)

    Li, Juan-Ying; Tang, Janet Yat Man; Jin, Ling; Escher, Beate I


    Bioavailable and bioaccessible fractions of sediment-associated contaminants are considered as better dose metrics for sediment-quality assessment than total concentrations. The authors applied exhaustive solvent extraction and nondepletive equilibrium sampling techniques to sediment samples collected along the Brisbane River in South East Queensland, Australia, which range from pristine environments to urban and industry-impacted areas. The wide range of chemicals expected prevents comprehensive chemical analysis, but a battery of cell-based bioassays sheds light on mixture effects of chemicals in relation to various modes of toxic action. Toxic effects were expressed as bioanalytical equivalent concentrations (BEQs) normalized to the organic carbon content of each sediment sample. Bioanalytical equivalent concentrations from exhaustive extraction agreed fairly well with values estimated from polydimethylsiloxane passive sampling extracts via the constant organic carbon to polydimethylsiloxane partition coefficient. Agreement was best for bioassays indicative of photosynthesis inhibition and oxidative stress response and discrepancy within a factor of 3 for the induction of the aryl hydrocarbon receptor. For nonspecific cytotoxicity, BEQ from exhaustive extraction were 1 order of magnitude higher than values from equilibrium sampling, possibly because of coextraction of bioactive natural organic matter that led to an overestimation of toxicity in the exhaustive extracts, which suggests that passive sampling is better suited in combination with bioanalytical assessment than exhaustive extraction.

  1. Marine invertebrate xenobiotic-activated nuclear receptors: their application as sensor elements in high-throughput bioassays for marine bioactive compounds. (United States)

    Richter, Ingrid; Fidler, Andrew E


    Developing high-throughput assays to screen marine extracts for bioactive compounds presents both conceptual and technical challenges. One major challenge is to develop assays that have well-grounded ecological and evolutionary rationales. In this review we propose that a specific group of ligand-activated transcription factors are particularly well-suited to act as sensors in such bioassays. More specifically, xenobiotic-activated nuclear receptors (XANRs) regulate transcription of genes involved in xenobiotic detoxification. XANR ligand-binding domains (LBDs) may adaptively evolve to bind those bioactive, and potentially toxic, compounds to which organisms are normally exposed to through their specific diets. A brief overview of the function and taxonomic distribution of both vertebrate and invertebrate XANRs is first provided. Proof-of-concept experiments are then described which confirm that a filter-feeding marine invertebrate XANR LBD is activated by marine bioactive compounds. We speculate that increasing access to marine invertebrate genome sequence data, in combination with the expression of functional recombinant marine invertebrate XANR LBDs, will facilitate the generation of high-throughput bioassays/biosensors of widely differing specificities, but all based on activation of XANR LBDs. Such assays may find application in screening marine extracts for bioactive compounds that could act as drug lead compounds.

  2. Analysis of Bioactive Components of Oilseed Cakes by High-Performance Thin-Layer Chromatography-(Bioassay Combined with Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Sue-Siang Teh


    Full Text Available Hemp, flax and canola seed cakes are byproducts of the plant oil extraction industry that have not received much attention in terms of their potential use for human food instead of animal feed. Thus, the bioactivity profiling of these oilseed cakes is of interest. For their effect-directed analysis, planar chromatography was combined with several (bioassays, namely 2,2-diphenyl-1-picrylhydrazyl scavenging, acetylcholine esterase inhibition, planar yeast estrogen screen, antimicrobial Bacillus subtilis and Aliivibrio fischeri assays. The streamlined high-performance thin-layer chromatography (HPTLC-bioassay method allowed the discovery of previously unknown bioactive compounds present in these oilseed cake extracts. In contrast to target analysis, the direct link to the effective compounds allowed comprehensive information with regard to selected effects. HPTLC-electrospray ionization-mass spectrometry via the elution-head based TLC-MS Interface was used for a first characterization of the unknown effective compounds. The demonstrated bioactivity profiling on the feed/food intake side may guide the isolation of active compounds for production of functional food or for justified motivation of functional feed/food supplements.

  3. Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay. (United States)

    Stark, Timo; Marxen, Sandra; Rütschle, Andrea; Lücking, Genia; Scherer, Siegfried; Ehling-Schulz, Monika; Hofmann, Thomas


    A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC-TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC-electrospray ionization (ESI)-TOF MS and the HPLC-ESI-MS/MS-SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

  4. Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays. (United States)

    Van der Linden, Sander C; Heringa, Minne B; Man, Hai-Yen; Sonneveld, Edwin; Puijker, Leo M; Brouwer, Abraham; Van der Burg, Bart


    It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity. Effluents from industry, hospital, and municipal sewage treatment plants, as well as tap water and different sources of surface water were tested. The CALUX reporter gene panel showed high sensitivity and specificity to known agonists, enabling discrimination between different receptor based endocrine responses present in the aquatic environment. Our results clearly showed the presence of agonistic activity on the ER, as well as on the AR, PR, and GR in the raw and wastewater and surface water extracts. However, no hormone receptor-mediated transactivation was detected in the drinking water or in the blank water. The levels of estrogenic activity were 0.2-0.5 ng E2-equiv/L for surface water and 0.4-1.0 ng E2-equiv/L for municipal effluents, which was consistent with previous studies. Surprisingly, the other hormonal activities were found to be present in similar or much higher levels. Most notably, glucocorticoid-like activity was detected in all samples, at surprisingly high levels ranging from 0.39-1.3 ng Dex-equiv/L in surface water and 11-243 ng Dex-equiv/L in effluents. When regarding the fact that dexamethasone in the GR CALUX bioassay is a factor 12 more potent than the natural hormone cortisol, results expressed as cortisol equivalents would range up to 2900 ng cortisol equiv/L. Further studies are needed to establish the identity of the active compounds and to

  5. Polyvinyl polypyrrolidone attenuates genotoxicity of silver nanoparticles synthesized via green route, tested in Lathyrus sativus L. root bioassay. (United States)

    Panda, Kamal K; Achary, V Mohan M; Phaomie, Ganngam; Sahu, Hrushi K; Parinandi, Narasimham L; Panda, Brahma B


    The silver nanoparticles (AgNPs) were synthesized extracellularly from silver nitrate (AgNO3) using kernel extract from ripe mango Mengifera indica L. under four different reaction conditions of the synthesis media such as the (i) absence of the reducing agent, trisodium citrate (AgNPI), (ii) presence of the reducing agent (AgNPII), (iii) presence of the cleansing agent, polyvinyl polypyrrolidone, PVPP (AgNPIII), and (iv) presence of the capping agent, polyvinyl pyrrolidone, PVP (AgNPIV). The synthesis of the AgNPs was monitored by UV-vis spectrophotometry. The AgNPs were characterised by the energy-dispersive X-ray spectroscopy, transmission electron microscopy, X-ray diffraction, and small-angle X-ray scattering. Functional groups on the AgNPs were established by the Fourier transform infrared spectroscopy. The AgNPs (AgNPI, AgNPII, AgNPIII and AgNPIV) were spherical in shape with the diameters and size distribution-widths of 14.0±5.4, 19.2±6.6, 18.8±6.6 and 44.6±13.2nm, respectively. Genotoxicity of the AgNPs at concentrations ranging from 1 to 100mgL(-1) was determined by the Lathyrus sativus L. root bioassay and several endpoint assays including the generation of reactive oxygen species and cell death, lipid peroxidation, mitotic index, chromosome aberrations (CA), micronucleus formation (MN), and DNA damage as determined by the Comet assay. The dose-dependent induction of genotoxicity of the silver ion (Ag(+)) and AgNPs was in the order Ag(+)>AgNPII>AgNPI>AgNPIV>AgNPIII that corresponded with their relative potencies of induction of DNA damage and oxidative stress. Furthermore, the findings underscored the CA and MN endpoint-based genotoxicity assay which demonstrated the genotoxicity of AgNPs at concentrations (≤10mgL(-1)) lower than that (≥10mgL(-1)) tested in the Comet assay. This study demonstrated the protective action of PVPP against the genotoxicity of AgNPIII which was independent of the size of the AgNPs in the L. sativus L. root bioassay

  6. The evaluation of dioxin and dioxin-like contaminants in selected food samples obtained from the Belgian market: comparison of TEQ measurements obtained through the CALUX bioassay with congener specific chemical analyses. (United States)

    Schoeters, Greet; Goyvaerts, Marie Pierre; Ooms, Daniëlla; Van Cleuvenbergen, Rudy


    A limited number of different foods were analysed for dioxin-like compounds by the CALUX bioassay which is an in vitro luciferase reporter gene assay measuring chemical activation of the aryl hydrocarbon receptor. Sixty-two milk samples were obtained from a surveillance campaign, 34 meat samples and 34 fishery products were purchased from the Belgian market. Bio-analytical and chemo-analytical dioxin toxicity equivalents (TEQ) values of the same milk samples were compared. Spearman's Rank correlation coefficients of 0.72, 0.67, 0.73 were obtained respectively between CALUX-TEQ and PCDD/F-TEQ, DL-PCB-TEQ and PCDD/F+DL-PCB-TEQ. The bioassay limit of detection was 0.1 pg TEQ from 1 g animal lipid, the limit of quantification was 0.4 pg TEQ. The repeatability of the CALUX bioassay (variability of butter fat samples analysed in the same run) showed a coefficient of variation (CV) of 10%, intra laboratory reproducibility based on independent runs of the same butter fat samples showed more variation (CV of 26% for samples above 2 pg TEQ/g lipid). All milk samples with a chemical TEQ value above the current limit value in Belgium showed an elevated CALUX-TEQ concentration, above 6 pg TEQ/g lipid. No false negative results were obtained. Based on the good correlation between CALUX-TEQ and chemically measured TEQ levels, the CALUX bioassay can be recommended as a screening tool for routine measurement of potentially toxic PHAHs in milk samples. Chemical analyses could then largely be restricted to positive samples, in order to identify the nature and to quantify the concentration of the chemicals that give the positive signal. Meat samples showed lower CALUX-TEQ values per gram lipid compared to fish samples. The fish samples showed a wider range of CALUX-TEQ values than the meat samples.

  7. A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays

    Directory of Open Access Journals (Sweden)

    Knols Bart GJ


    Full Text Available Abstract Background Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared. Methods Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes. Results K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time. Conclusions K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers

  8. Guidance document for prepermit bioassay testing of low-level radioactive waste

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, S.L.; Harrison, F.L.


    In response to the mandate of Public Law 92-532, the Marine Protection, Research, and Sanctuaries Act (MPRSA) of 1972, as amended, the Environmental Protection Agency (EPA) has developed a program to promulgate regulations and criteria to control the ocean disposal of radioactive wastes. The EPA seeks to understand the mechanisms for biological response of marine organisms to the low levels of radioactivity that may arise from the release of these wastes as a result of ocean-disposal practices. Such information will play an important role in determining the adequacy of environmental assessments provided to the EPA in support of any disposal permit application. Although the EPA requires packaging of low-level radioactive waste to prevent release during radiodecay of the materials, some release of radioactive material into the deep-sea environment may occur when a package deteriorates. Therefore, methods for evaluating the impact on biota are being evaluated. Mortality and phenotypic responses are not anticipated at the expected low environmental levels that might occur if radioactive materials were released from the low-level waste packages. Therefore, traditional bioassay systems are unsuitable for assessing sublethal effects on biota in the marine environment. The EPA Office of Radiation Programs (ORP) has had an ongoing program to examine sublethal responses to radiation at the cellular level, using cytogenetic end points. This technical guidance report represents prepermit bioassay procedures that potentially may be applicable to the assessment of effects from a mixture of radionuclides that could be released from a point source at the ocean bottom. Methodologies along with rationale and a discussion of uncertainty are presented for the sediment benthic bioassay protocols identified in this report.

  9. Comparison between Two Bioassays in Monitoring and Evaluation of Water Quality%两种生物测试法在水质监测与评价中的应用比较

    Institute of Scientific and Technical Information of China (English)

    徐亚平; 刘凤枝; 刘岩; 邵晓龙


    [ Objective] The aim was to compare the application of two bioassays to monitoring and evaluation of water quality. [ Method] By using two bioassays (micronucleus detecting technology based on Vicia faba root tip and the bioassay by luminous bacteria) , we monitored and evaluated seven kinds of single solutions added different pollutants (Hg, Cd, As, Cr6+ , Pb, LAS and CODCr) and a mixed solution added those seven pollutants. Afterwards, we compared their results under the same pollutant and concentration, so as to study the two bioassays' sensitivity and sensitive concentration to the seven pollutants. [Result] Under the same pollutant and concentration, micronucleus detecting technology based on Vicia faba root tip was response to Hg, Cd, As and Cr 6+ , but wasn' t response to Pb, LAS and CODCr. However, the bioassay by luminous bacteria was response to most pollutants except Cr6+. Comparing the sensitive degree and concentration to each pollutant, they can complement each other. For these seven pollutants, bioassay by luminous bacteria was better than micronucleus detecting technology based on Vicia faba root tip. Meanwhile, from the testing result of the mixed solution, the combined toxicity of several pollutants in lower concentrations was serious. At the same time, contrasted to normal chemistry methods, bioassays were fast and effective. [ Conclusion ] The research could provide theoretical references for the correlation study of bioassays.%[目的]比较两种生物测试法在水质监测与评价中的应用.[方法]分别采用蚕豆根尖细胞微核法和发光细菌法两种常用生物测试方法,测试了不同浓度梯度的重金属(汞、镉、砷、六价铬、铅)、有机污染物(阴离子表面活性剂、化学需氧量)溶液及其混合溶液,比较这两种方法在相同污染物和污染浓度下的检测结果,以了解其各自对常见污染物的敏感程度及敏感浓度.[结果]在相同的污染物及浓度梯度下,蚕豆根

  10. Shell growth of unfed oysters in the laboratory: a sublethal bioassay system for pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Conger, K.A.; Swift, M.L.; Reeves, J.B. III; Lakshmanan, S.


    Unfed oysters, Crassostrea virginica Gmelin, in 12 g/l commercial grade artificial sea-water supplemented with calcium bicarbonate (approximately 7 mM Ca/sup 2 +/ and HCO/sub 3//sup -/) deposit shell for four to six weeks. A no-growth critical calcium ion concentration of 1.5 mM was determined in this study. A simple sublethal bioassay system can be developed utilizing the observed shell growth. Significant (p < 0.001) inhibition of shell deposition in oysters subjected to an initial concentration of 0.25 mg Cd/sup 2 +//l demonstrates the efficacy of the proposed method.

  11. Identification and bioassay of sex pheromone components of carob moth,Ectomyelois ceratoniae (Zeller). (United States)

    Baker, T C; Francke, W; Millar, J G; Löfstedt, C; Hansson, B; Du, J W; Phelan, P L; Vetter, R S; Youngman, R; Todd, J L


    Three sex pheromone components of the carob moth were isolated and identified from the extract of female pheromone glands, using a variety of techniques including coupled gas chromatographic-electroantennographic recordings, coupled gas chromatographic-mass spectrometric analysis, microozonolysis, electroantennographic assays of monounsaturated standards, wind-tunnel bioassays, and field trials. The major component was identified as (Z,E)-9,11,13-tetradecatrienal, a novel lepidopterous pheromone component structure. Two minor components, either one of which improves the upwind flight response of males when blended with the major component, were identified as (Z,E)-9,11-tetradecadienal, and (Z)-9-tetra-decenal.

  12. Promising Aedes aegypti repellent chemotypes identified through integrated QSAR, virtual screening, synthesis, and bioassay. (United States)

    Oliferenko, Polina V; Oliferenko, Alexander A; Poda, Gennadiy I; Osolodkin, Dmitry I; Pillai, Girinath G; Bernier, Ulrich R; Tsikolia, Maia; Agramonte, Natasha M; Clark, Gary G; Linthicum, Kenneth J; Katritzky, Alan R


    Molecular field topology analysis, scaffold hopping, and molecular docking were used as complementary computational tools for the design of repellents for Aedes aegypti, the insect vector for yellow fever, chikungunya, and dengue fever. A large number of analogues were evaluated by virtual screening with Glide molecular docking software. This produced several dozen hits that were either synthesized or procured from commercial sources. Analysis of these compounds by a repellent bioassay resulted in a few highly active chemicals (in terms of minimum effective dosage) as viable candidates for further hit-to-lead and lead optimization effort.

  13. Bioassay-guided isolation and identification of an antibacterial compound from Ferula persica var. persica roots

    Directory of Open Access Journals (Sweden)

    Ahmad-Reza Shahverdi


    Full Text Available The antibacterial activities of the chloroform and water extracts of Ferula persica var. persica (Apiaceaeroots were studied by the disk diffusion method. While the chloroform extract of F. persica roots showed antibacterial activity, the water extract of the roots at the concentrations that tested did not show any activity. By bioassay-guided fractionation of the chloroform extract of the roots by preparative thin layer chromatography (PTLC a compound was found which was active against some bacteria. By conventional spectroscopy methods the active fraction was identified as umbelliprenin. This coumarin was mostly active against B. subtillis, B. cereus, E. coli, K. ponumoniae, S. typhi, S. aureus, and S. epidermilis.

  14. Test System Stability and Natural Variability of a Lemna Gibba L. Bioassay (United States)

    Scherr, Claudia; Simon, Meinhard; Spranger, Jörg; Baumgartner, Stephan


    Background In ecotoxicological and environmental studies Lemna spp. are used as test organisms due to their small size, rapid predominantly vegetative reproduction, easy handling and high sensitivity to various chemicals. However, there is not much information available concerning spatial and temporal stability of experimental set-ups used for Lemna bioassays, though this is essential for interpretation and reliability of results. We therefore investigated stability and natural variability of a Lemna gibba bioassay assessing area-related and frond number-related growth rates under controlled laboratory conditions over about one year. Methology/Principal Findings Lemna gibba L. was grown in beakers with Steinberg medium for one week. Area-related and frond number-related growth rates (r(area) and r(num)) were determined with a non-destructive image processing system. To assess inter-experimental stability, 35 independent experiments were performed with 10 beakers each in the course of one year. We observed changes in growth rates by a factor of two over time. These did not correlate well with temperature or relative humidity in the growth chamber. In order to assess intra-experimental stability, we analysed six systematic negative control experiments (nontoxicant tests) with 96 replicate beakers each. Evaluation showed that the chosen experimental set-up was stable and did not produce false positive results. The coefficient of variation was lower for r(area) (2.99%) than for r(num) (4.27%). Conclusions/Significance It is hypothesised that the variations in growth rates over time under controlled conditions are partly due to endogenic periodicities in Lemna gibba. The relevance of these variations for toxicity investigations should be investigated more closely. Area-related growth rate seems to be more precise as non-destructive calculation parameter than number-related growth rate. Furthermore, we propose two new validity criteria for Lemna gibba bioassays

  15. Bioassay-guided isolation of apigenin with GABA-benzodiazepine activity from Tanacetum parthenium

    DEFF Research Database (Denmark)

    Jäger, Anna Katharina; Krydsfeldt, Katrine; Rasmussen, Hasse Bonde


    Extracts of Tanacetum parthenium are used in the prophylactic treatment of migraine and have also been used in Danish folk medicine for the treatment of epilepsy. An ethanol extract of T. parthenium showed high affinity for the GABA(A)-benzodiazepine site. An ethanol extract of T. parthenium...... was fractionated by VLC on silica and preparative C18 HPLC. Each step was monitored with the GABA(A)-benzodiazepine bioassay. The fractionation led to the isolation of apigenin, which may be responsible for CNS-effects of T. parthenium extracts. Copyright (c) 2009 John Wiley & Sons, Ltd....

  16. Ecotoxicological classification of the Berlin river system using bioassays in respect to the European Water Framework Directive. (United States)

    Huschek, Gerd; Hansen, P-D


    Bioassays as well as biochemical responses (biomarkers) in ecosystems due to environmental stress provide us with signals (environmentally signalling) of potential damage in the environment. If these responses are perceived in this early stage in ecosystems, the eventual damage can be prevented. Once ecosystem damage has occurred, the remedial action processes for recovery could be expensive and pose certain logistical problems. Ideally, "early warning signals" in ecosystems using sensing systems of biochemical responses (biomarkers) would not only tell us the initial levels of damage, but these signals will also provide us with answers by the development of control strategies and precautionary measures in respect to the European Water Framework Directive (WFD). Clear technical guidelines or technical specifications on monitoring are necessary to establish and characterise reference conditions for use in an ecological status classification system for surface water bodies. For the Ecotoxicological Risk Assessment (ERA) of endocrine effects we used an approach of the exposure - dose - response concept. Based on the "Ecototoxicological Classification System of Sediments" that uses pT-values to classify effects in different river systems, we transferred the bio-monitoring data to the five-level ecological system of the WFD. To understand the complexity of the structure of populations and processes behind the health of populations, communities and ecosystems an ERA should establish links between natural factors, chemicals, and biological responses so as to assess causality. So, our ecological monitoring assessment has incorporated exposure & effects data.

  17. Determination of estrogenic activity in the river Chienti (Marche Region, Italy) by using in vivo and in vitro bioassays. (United States)

    Cocci, Paolo; Palermo, Francesco Alessandro; Quassinti, Luana; Bramucci, Massimo; Miano, Antonino; Mosconi, Gilberto


    Environmental estrogen-like compounds (i.e. xenoestrogens) are a variety of pollutants, ranging from synthetic to natural occurring molecules, that are found in surface and waste waters over a wide range of concentrations. In aquatic environment, the overall estrogenic activity is often due to the presence of a mixture of chemicals and their degraded products which can induce synergistic effects. Current strategies for monitoring estrogen-like chemicals are based on the use of a battery of in vivo and in vitro ecotoxicological tests. In this regard, the aim of the present work was to carry out a bio-monitoring study for testing estrogenicity of the Chienti river (Marche Region, Italy) by using both an E-screen and a vitellogenin (Vtg) induction assay in juvenile goldfish. Three sites were used for analysis, localized at the mouth (sampling point 1), in the middle (sampling point 2) and at the origin (sampling point 3) of Chienti river. For most of the water samples (i.e. samples collected at sampling points 2 and 3), clear estrogenic activity was detected in the E-screen assay suggesting different proliferation activities in function of the collecting site. In contrast, the Vtg ELISA demonstrated that water samples collected from each sampling point were estrogenic. Overall, we showed for the first time that the estrogenic activities in water samples from the Chienti river were significant in both in vivo and in vitro; we also observed a different sensitivity between bioassays.

  18. Bioassays for assessing jasmonate-dependent defenses triggered by pathogens, herbivorous insects, or beneficial rhizobacteria. (United States)

    Van Wees, Saskia C M; Van Pelt, Johan A; Bakker, Peter A H M; Pieterse, Corné M J


    Jasmonates, together with other plant hormones, are important orchestrators of the plant immune system. The different hormone-controlled signaling pathways cross-communicate in an antagonistic or a synergistic manner, providing the plant with a powerful capacity to finely regulate its immune response. Jasmonic acid (JA) signaling is required for plant resistance to harmful organisms, such as necrotrophic pathogens and herbivorous insects. Furthermore, JA signaling is essential in interactions of plants with beneficial microbes that induce systemic resistance to pathogens and insects. The role of JA signaling components in plant immunity can be studied by performing bioassays with different interacting organisms. Determination of the level of resistance and the induction of defense responses in plants with altered JA components, through mutation or ectopic expression, will unveil novel mechanisms of JA signaling. We provide detailed protocols of bioassays with the model plant Arabidopsis thaliana challenged with the pathogens Botrytis cinerea and Pseudomonas syringae, the insect herbivore Pieris rapae, and the beneficial microbe Pseudomonas fluorescens. In addition, we describe pharmacological assays to study the modulation of JA-regulated responses by exogenous application of combinations of hormones, because a simultaneous rise in hormone levels occurs during interaction of plants with other organisms.

  19. A battery of bioassays for the evaluation of phenanthrene biotoxicity in soil. (United States)

    Khan, Muhammad Imran; Cheema, Sardar Alam; Tang, Xianjin; Hashmi, Muhammad Zaffar; Shen, Chaofeng; Park, Joonhong; Chen, Yingxu


    A battery of bioassays was used to assess the ecotoxicological risk of soil spiked with a range of phenanthrene levels (0.95, 6.29, 38.5, 58.7, 122, and 303 μg g(-1) dry soil) and aged for 69 days. Multiple species (viz. Brassica rapa, Eisenia feotida, Vibrio fischeri), representing different trophic levels, were used as bioindicator organisms. Among acute toxicity assays tested, the V. fischeri luminescence inhibition assay was the most sensitive indicator of phenanthrene biotoxicity. More than 15 % light inhibition was found at the lowest phenanthrene level (0.95 μg g(-1)). Furthermore, comet assay using E. fetida was applied to assess genotoxicity of phenanthrene. The strong correlation (r (2) ≥ 0.94) between phenanthrene concentration and DNA damage indicated that comet assay is appropriate for testing the genotoxic effects of phenanthrene-contaminated soil. In the light of these results, we conclude that the Microtox test and comet assay are robust and sensitive bioassays to be employed for the risk evaluation of polycyclic aromatic hydrocarbon-contaminated soil.

  20. Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban (United States)

    Torres, Tiago; Cunha, Isabel; Martins, Rosário; Santos, Miguel M.


    Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants. PMID:27775672

  1. DOSEXPRT: A bioassay dosimetry code for Martin Marietta Energy Systems, Inc.

    Energy Technology Data Exchange (ETDEWEB)

    Ward, R.C.; Eckerman, K.F.


    The bioassay code DOSEXPRT was developed for Martin Marietta Energy Systems, Inc., to provide compliance with Department of Energy (DOE) Order 5480, Chapter 11. DOSEXPRT computes the intake of a radionuclide in any year (considering both acute and chronic intakes) from in vivo measurements of the retained activity and/or measurements of the activity in excreta. The committed effective and organ doses for the intake are computed as well as the effective and organ doses expected to be received in each calendar year out to 50 years beyond the year of intake. The bioassay records used as input for DOSEXPRT are extracted from the Martin Marietta Energy Systems Occupational Health Information System (OHIS). DOSEXPRT implements a set of algorithms with parameters governing the translocation, retention, and excretion of the nuclide contained in data files specific to the nuclide. These files also contain dose-per-unit-intake coefficients used to compute the committed dose equivalent for the intakes in the year. Annual organ and effective doses are computed using additional dose-rate files that contain data on the dose rate at various times following a unit intake. If measurements are presented for more than one assay for a given nuclide, DOSEXPRT estimates the intake by applying weights assigned in the nuclide file for each assay. DOSEXPRT is accessed off the OHIS MENU No. 4 and designed to be run as a batch processor, but can also be run interactively for testing purposes.

  2. DOSEXPRT: A bioassay dosimetry code for Martin Marietta Energy Systems, Inc

    Energy Technology Data Exchange (ETDEWEB)

    Ward, R.C.; Eckerman, K.F.


    The bioassay code DOSEXPRT was developed for Martin Marietta Energy Systems, Inc., to provide compliance with Department of Energy (DOE) Order 5480, Chapter 11. DOSEXPRT computes the intake of a radionuclide in any year (considering both acute and chronic intakes) from in vivo measurements of the retained activity and/or measurements of the activity in excreta. The committed effective and organ doses for the intake are computed as well as the effective and organ doses expected to be received in each calendar year out to 50 years beyond the year of intake. The bioassay records used as input for DOSEXPRT are extracted from the Martin Marietta Energy Systems Occupational Health Information System (OHIS). DOSEXPRT implements a set of algorithms with parameters governing the translocation, retention, and excretion of the nuclide contained in data files specific to the nuclide. These files also contain dose-per-unit-intake coefficients used to compute the committed dose equivalent for the intakes in the year. Annual organ and effective doses are computed using additional dose-rate files that contain data on the dose rate at various times following a unit intake. If measurements are presented for more than one assay for a given nuclide, DOSEXPRT estimates the intake by applying weights assigned in the nuclide file for each assay. DOSEXPRT is accessed off the OHIS MENU No. 4 and designed to be run as a batch processor, but can also be run interactively for testing purposes.

  3. Estimation of cytotoxic potency by brine shrimp lethality bioassay application of Clerodendrum infortunatum Linn.

    Institute of Scientific and Technical Information of China (English)

    Talukdar Muhammad Waliullah; Akter MstYeasmin; Ashraful MdAlam; Wahedul Md Islam; Parvez Hassan


    Objective: To learn a scientific and systematic knowledge of anticancer, antimicrobial and pharmacological activities of natural products and estimate cytotoxic potency by using ethanol and chloroform extracts of root, leaf and stem of Clerodendrum infortunatum (Verbenaceae) due to its random use in customary and traditional medicine to cure common ailments such as intestinal disorder, diarrhea, tuberculosis and respiratory problems etc. Methods: The in vitro application was carried out with the bench-top bioassay method by using brine shrimp lethality bioassay. Results: All of the crude extracts were found to be lethal and effective. The LC50 value of ethyl alcohol fraction of root was 20.845 mg/L compared to the standard drug tetracycline of 14.675 mg/L to brine shrimp nauplii, indicating that the extracts were biologically active. Conclusions: The cytotoxic study of LC50 value showed that a good correlation with the antibiotic tetracycline. From the comparative correlation error bars and percentage, we understood that ethyl alcohol fraction of root extract was very effective. This study serves as a basis for further research to lead compounds to be isolated so that it may be as a template for the implications of these results for bioactivity and drug discovery potential of herbal products.

  4. The Tradescantia pallida var. purpurea active bioassay for water monitoring: evaluating and comparing methodological conditions

    Directory of Open Access Journals (Sweden)

    Mara Betânia Brizola Cassanego


    Full Text Available Tradescantia pallida var. purpurea cuttings with flower buds are utilized in bioassays to diagnose genotoxic effects of water. The literature describes different substances used to adapt and recover the cuttings before and after exposure to water samples and also describes the effects of different exposure times. This study evaluated and compared the micronuclei (MCN frequencies in T. pallida when cuttings with flower buds were submitted to different methodological conditions. The bioassay was then applied bimonthly during seven months to assess the genotoxic potential of a site located on the Sinos River in Campo Bom, Rio Grande do Sul, Brazil. Micronuclei frequencies in buds of cuttings adapted and recovered in distilled water and in Hoagland solution were 3.0 and 2.9, respectively, for cuttings exposed to river water, and 1.19 and 1.23 in controls. No significant differences among MCN frequencies were observed when cuttings were exposed for 8, 24 or 32 hours to river water (from 3.07 to 4.73 and in controls (from 1.13 to 2.00 in all samplings during a year. Adaptation and recovery of cuttings in distilled water or Hoagland solution and exposure for different times did not influence the response of T. pallida, indicating that all the conditions tested are viable for biomonitoring of water genotoxicity. Water samples from the Sinos River presented genotoxicity during the period monitored, evidenced by the MCN frequencies recorded which were significantly higher than the frequencies of the controls.

  5. High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity. (United States)

    Bechstein, Daniel J B; Lee, Jung-Rok; Ooi, Chin Chun; Gani, Adi W; Kim, Kyunglok; Wilson, Robert J; Wang, Shan X


    Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles' biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms.

  6. Pharmacodynamics of TRPV1 Agonists in a Bioassay Using Human PC-3 Cells

    Directory of Open Access Journals (Sweden)

    Daniel Alvarez-Berdugo


    Full Text Available Purpose. TRPV1 is a multimodal channel mainly expressed in sensory neurons. We aimed to explore the pharmacodynamics of the TRPV1 agonists, capsaicin, natural capsaicinoids, and piperine in an in vitro bioassay using human PC-3 cells and to examine desensitization and the effect of the specific antagonist SB366791. Methods. PC-3 cells expressing TRPV1 were incubated with Fluo-4. Fluorescence emission changes following exposition to agonists with and without preincubation with antagonists were assessed and referred to maximal fluorescence following the addition of ionomycin. Concentration-response curves were fitted to the Hill equation. Results. Capsaicin and piperine had similar pharmacodynamics (Emax 204.8 ± 184.3% piperine versus 176.6 ± 35.83% capsaicin, P=0.8814, Hill coefficient 0.70 ± 0.50 piperine versus 1.59 ± 0.86 capsaicin, P=0.3752. In contrast, capsaicinoids had lower Emax (40.99 ± 6.14% capsaicinoids versus 176.6 ± 35.83% capsaicin, P<0.001. All the TRPV1 agonists showed significant desensitization after the second exposition and their effects were strongly inhibited by SB366791. Conclusion. TRPV1 receptor is successfully stimulated by capsaicin, piperine, and natural capsaicinoids. These agonists present desensitization and their effect is significantly reduced by a TRPV1-specific antagonist. In addition, PC-3 cell bioassays proved useful in the study of TRPV1 pharmacodynamics.

  7. Tradescantia-micronucleus (Trad-MCN) bioassay on clastogenicity of wastewater and in situ monitoring. (United States)

    Ruiz, E F; Rabago, V M; Lecona, S U; Perez, A B; Ma, T H


    The Tradescantia-micronucleus (Trad-MCN) bioassay was used to determine the clastogenicity of wastewater samples collected from the Arena canal which contains effluent from the industrial district Benito Juarez of the city of Queretaro, Mexico. Fifteen wastewater samples which were collected, in most cases, at bi-weekly intervals beginning in September 1986 through February 1988, after a 3-fold dilution were used to treat Tradescantia plant cuttings. The clastogenicity expressed in terms of micronucleus frequencies of treated groups (30 h of treatment without recovery time) was significantly (0.01) higher than that of the tapwater control groups. The Trad-MCN bioassay was also used for in situ monitoring of air pollutants for the clastogenicity at 3 sites near the industrial and residential areas (Flores Magon, Conalep and Bellas Artes) of the city of Queretaro. Fourteen monitoring trips were made to each of the 3 sites at monthly intervals beginning in May 1988 through June 1990. Seasonal variation of micronucleus frequencies was exhibited with the peak clastogenicities shown in May and June 1988, June 1989 and April 1990 at the three sites. Micronucleus frequencies of all the exposed groups at the Conalep site, a predominantly industrial area, were markedly higher than that of the laboratory control groups throughout the 2-year period.

  8. Evaluation of bioassays versus contaminant concentrations in explaining the macroinvertebrate community structure in the Rhine-Meuse delta, The Netherlands. (United States)

    Peeters, E T; Dewitte, A; Koelmans, A A; van der Velden, J A; den Besten, P J


    It is often assumed that bioassays are better descriptors of sediment toxicity than toxicant concentrations and that ecological factors are more important than toxicants in structuring macroinvertebrate communities. In the period 1992 to 1995, data were collected in the enclosed Rhine-Meuse delta, The Netherlands, on macroinvertebrates, sediment toxicity, sediment contaminant concentrations, and ecological factors. The effect of various groups of pollutants (polycyclic aromatic hydrocarbons, trace metals, oil, polychlorinated biphenyls) and of ecological variables on the structure of the macroinvertebrate community were quantified. Ecological factors explained 17.3% of the macroinvertebrate variation, while contaminants explained 13.8%. Another 14.7% was explained by the covariation between ecological variables and contaminants. Polycyclic aromatic hydrocarbons explained a larger part of the variation than trace metals. The contributions of oil and polychlorinated biphenyls were small but significant. Elevated contaminant concentrations were significantly associated with differences in the macroinvertebrate food web structure. The response in bioassays (Vibrio fischeri, Daphnia magna, Chironomus riparius) was susceptible to certain contaminants but also to certain ecological factors. There was a weak correlation between in situ species composition and bioassays; 1.9% of in situ macroinvertebrate variation was explained by the bioassay responses. This seems to contradict the validity of using bioassays for a system-oriented risk assessment. Possible reasons for this discrepancy might be the manipulations of the sediment before the test and a higher pollutant tolerance of the in situ macroinvertebrates. Thus, macroinvertebrate field surveys and laboratory bioassays yield different types of information on ecotoxicological effects, and both are recommended in sediment risk assessment procedures.

  9. Functional bioassays for immune monitoring of high-risk neuroblastoma patients treated with ch14.18/CHO anti-GD2 antibody.

    Directory of Open Access Journals (Sweden)

    Nikolai Siebert

    Full Text Available Effective treatment of high-risk neuroblastoma (NB remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC and antibody-dependent cellular cytotoxicity (ADCC were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T cell ratio of 20:1 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40:1. Optimal results for CDC were found with a serum dilution at 1:8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV were below 20%. Sample quality following storage at room temperature (RT showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m(2 revealed GD2-specific increases in CDC (4.5-9.4 fold and ADCC (4.6-6.0 fold on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.

  10. Establishment of quantitative sequencing and filter contact vial bioassay for monitoring pyrethroid resistance in the common bed bug, Cimex lectularius. (United States)

    Seong, Keon Mook; Lee, Da-Young; Yoon, Kyong Sup; Kwon, Deok Ho; Kim, Heung Chul; Klein, Terry A; Clark, J Marshall; Lee, Si Hyeock


    Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel alpha-subunit gene have been identified in deltamethrin-resistant bed bugs. A quantitative sequencing (QS) protocol was developed to establish a population-based genotyping method as a molecular resistance-monitoring tool based on the frequency of the two mutations. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.9928). As determined by QS, neither mutation was found in a bed bug population collected in 1993. Populations collected in recent years (2007-2009), however, exhibited completely or nearly saturating L925I mutation frequencies and highly variable frequencies of the V419L mutation. In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to deltamethrin and lambda-cyhalothrin. A pyrethroid-resistant strain showed >9,375- and 6,990-fold resistance to deltamethrin and lambda-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB should greatly facilitate the detection and monitoring of pyrethroid-resistant bed bugs in the field. The advantages of FCVB as an on-site resistance-monitoring tool are discussed.

  11. In vivo genotoxicity of furan in F344 rats at cancer bioassay doses

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Wei, E-mail: [Division of Genetic and Molecular Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Petibone, Dayton M. [Division of Genetic and Molecular Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Latendresse, John R. [Toxicologic Pathology Associates, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Pearce, Mason G. [Division of Genetic and Molecular Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Muskhelishvili, Levan; White, Gene A. [Toxicologic Pathology Associates, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Chang, Ching-Wei [Division of Personalized Nutrition and Medicine, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Mittelstaedt, Roberta A.; Shaddock, Joseph G.; McDaniel, Lea P. [Division of Genetic and Molecular Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Doerge, Daniel R. [Division of Biochemical Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States); Morris, Suzanne M.; Bishop, Michelle E.; Manjanatha, Mugimane G.; Aidoo, Anane; Heflich, Robert H. [Division of Genetic and Molecular Toxicology, US FDA/National Center for Toxicological Research, Jefferson, AR 72079 (United States)


    Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8 mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16 mg/kg. Rats were killed 3 h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity. -- Highlights: ► Furan is a potent rodent liver carcinogen and represents a human cancer risk. ► Furan induces DNA damage in rat liver at cancer bioassay doses. ► Furan induces oxidative stress, inflammation and cell proliferation in rat liver. ► Expression of

  12. Contaminated sediments and bioassay responses of three macroinvertebrates, the midge larva Chironomus riparius, the water louse Asellus aquaticus and the mayfly nymph Ephoron virgo

    NARCIS (Netherlands)

    Lange, de H.J.; Haas, de E.M.; Maas, H.; Peeters, E.T.H.M.


    Bioassays are widely used to estimate ecological risks of contaminated sediments. We compared the results of three whole sediment bioassays, using the midge larva Chironomus riparius, the water louse Asellus aquaticus, and the mayfly nymph Ephoron virgo. We used sediments from sixteen locations in t

  13. Effects of conidial densities and spray volume of Metarhizium anisopliae and Beauveria bassiana fungal suspensions on conidial viability, droplet size and deposition coverage in bioassay using a novel bioassay spray system (United States)

    Experiments were conducted to study the conidial viability during bioassay spray with different suspensions of Metarhizium anisopliae ATCC 62176 and Beauveria bassiana NI8, and to investigate the effects of conidial density and spray volume on the distribution of droplet size and deposit coverage us...

  14. Multi-scale magnetic nanoparticle based optomagnetic bioassay for sensitive DNA and bacteria detection

    DEFF Research Database (Denmark)

    Tian, Bo; Zardán Gómez De La Torre, Teresa; Donolato, Marco


    for point-of-care diagnostics, utilizing functionalized magnetic nanoparticles (100 nm) with Brownian relaxation behavior is optimized in order to obtain higher detection sensitivity for DNA molecules and bacteria. Presence of target DNA sequences or bacteria changes the dynamic behavior of the magnetic...... nanoparticles (binding to the target) and thus the optomagnetic response of the sample, which is measured by an optomagnetic setup including a 405 nm laser and a photodetector. The limit of detection is mainly set by the lowest measurable concentration of magnetic nanoparticles. Herein, as new results compared...... to previous work, we systematically optimize the concentration of 100 nm magnetic nanoparticles to increase the assay sensitivity and lower the limit of detection. To enable biplex detection, we perform this optimization in the presence of larger 250 nm magnetic nanoparticles that do not interact...

  15. A novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis. (United States)

    Aytur, Turgut; Foley, Jonathan; Anwar, Mekhail; Boser, Bernhard; Harris, Eva; Beatty, P Robert


    New technologies are greatly needed to improve laboratory tests that can be used in point-of-care clinical settings. Here, a biosensor was used to detect micron-scale paramagnetic beads in order to replace the conventional enzymatic label used in ELISAs. This novel biosensor was fabricated through standard complementary metal oxide semiconductor (CMOS) manufacturing and was used to quantify magnetic beads bound to the sensor surface by immunological recognition, analogous to ELISA. CMOS technology can integrate multiple laboratory functions into the sensor chip, potentially enabling inexpensive, compact and sophisticated diagnostic systems for a number of diseases. We present results for two immunological assays: antigen capture of purified mouse IgG and detection of human anti-dengue virus IgG in clinical serum samples. The sensitivity of detecting purified protein with magnetic beads was comparable to ELISA. We found a high correlation between the ELISA optical density and the biosensor output in the clinical assay. We also demonstrate the use of a controlled magnetic field to remove non-specifically bound magnetic beads from the sensor surface, effectively washing the sensor surface. This novel sensor can be mass-produced at low cost and can detect magnetic beads bound to the surface through specific antibody-antigen interactions, making it a potential platform for new simplified and rapid point-of-care diagnostic tests.

  16. A chromosomally based luminescent bioassay for mercury detection in red soil of China

    Energy Technology Data Exchange (ETDEWEB)

    Wei, He [Chinese Academy of Sciences, Nanking (China). State Key Lab. of Soil and Sustainable Agriculture; Nanjing Normal Univ., Nanking (China). College of Life Science; Cheng, Han; Ting, Mao; Zhong, Wen-Hui [Nanjing Normal Univ., Nanking (China). College of Chemistry and Environmental Science; Lin, Xian-Gui [Chinese Academy of Sciences, Nanking (China). State Key Lab. of Soil and Sustainable Agriculture


    A luminescent reporter gene system was constructed by fusing the mercury-inducible promoter, P{sub merT}, and its regulatory gene, merR, with a promoterless reporter gene EGFP. A stable and nonantibiotic whole-cell reporter (BMB-ME) was created by introducing the system cassette into the chromosome of Pseudomonas putida strain and then applied it for mercury detection in the red soil of China. Spiked with 10 and 100 {mu}g g{sup -1} Hg{sup 2+} and after 15 and 30 days incubation, soil samples were extracted and evaluated water soluble, bioavailable, organic matter bound, and residual fractions of mercury by both BMB-ME and chemical way. The expression of EGFP was confirmed in soil extraction, and fluorescence intensity was quantified by luminescence spectrometer. The sensor strain BMB-ME appeared to have a detection range similar to that of reversed-phase high-performance liquid chromatography method. The optimal temperature for EGFP expression was 35 C and the lowest detectable concentration of Hg{sup 2+} 200 nM. Cu{sup 2+}, Fe{sup 2+}, Mn{sup 2+}, Sn{sup 2+}, Zn{sup 2+}, Co{sup 2+}, Ag{sup +}, Ba{sup 2+}, Mg{sup 2+}, and Pb{sup 2+} ions at nanomolar level did not interfere with the measurement. These results showed that the BMB-ME constitute an adaptable system for easy sensing of small amounts of mercury in the red soil of China. (orig.)

  17. SERMs and SARMs: Detection of their activities with yeast based bioassays

    NARCIS (Netherlands)

    Bovee, T.F.H.; Thevis, M.; Hamers, A.R.M.; Peijnenburg, A.A.C.M.; Nielen, M.W.F.; Schoonen, W.G.E.J.


    Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Many of these compounds will find their use in therapeutic treatments. However, they also

  18. Rapid, Bioassay-Guided Process for the Detection and Identification of Antibacterial Neem Oil Compounds. (United States)

    Krüzselyi, Dániel; Nagy, Róbert; Ott, Péter G; Móricz, Ágnes M


    Bioassay guidance was used along the whole process including method development, isolation and identification of antibacterial neem (Azadirachta indica) oil compounds. The biomonitoring was performed by direct bioautography (DB), a combination of thin-layer chromatography (TLC) and antimicrobial detection. DB of neem oil showed one antibacterial zone that was not UV-active; therefore, the TLC separation was improved under DB control. The chromatographic zone that exhibited activity against Bacillus subtilis, Xanthomonas euvesicatoria, Aliivibrio fischeri, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was characterized by TLC reagents, indicating a lipophilic, fatty acid-like chemical feature. Two compounds were found and identified in the active zone by high-performance liquid chromatography-electrospray ionization mass spectrometry as linoleic and oleic acids. Both fatty acids inhibited B. subtilis, but A. fischeri was sensitive only against linoleic acid.

  19. Respiratory tract clearance model for dosimetry and bioassay of inhaled radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, M.R.; Birchall, A. (National Radiological Protection Board, Chilton (UK)); Cuddihy, R.G. (Inhalation Toxicology Research Inst., Albuquerque, NM (USA)); James, A.C. (Pacific Northwest Lab., Richland, WA (USA)); Roy, M. (CEA Centre d' Etudes Nucleaires de Fontenay-aux-Roses, 92 (France). Inst. de Protection et de Surete Nucleaire)


    The ICRP Task Group on Respiratory Tract Models is developing a model to describe the retention and clearance of deposited radionuclides for dose-intake calculations and interpretation of bioassay data. Clearance from each region is treated as competition between mechanical transport, which moves particles to the gastro-intestinal tract and lymph nodes, and the translocation of material to blood. It is assumed that mechanical transport rates are the same for all materials, and that rates of translocation to blood are the same in all regions. Time-dependent clearance is represented by combinations of compartments. Representative values of parameters to describe mechanical transport from the human respiratory tract have been estimated, and guidance is given on the determination of translocation rates. It is emphasized that the current version of the model described here is still provisional. 30 refs.

  20. Symposium on Short-Term Genetic Bioassays in the Evaluation of Complex Environmental Mixtures

    CERN Document Server

    Sandhu, Shahbeg; Lewtas, Joellen; Claxton, Larry; Strauss, Gary; Nesnow, Stephen


    With this proceedings of the fourth symposium on complex mixtures, we continue to revise and extend our knowledge of genetic methods for the evaluation of chemical mixtures in the environment. The early chapters of this volume are devoted to new bioassay techniques that are directly applicable to the monitoring of environments contaminated with genotoxic chemicals. Microbiological methods have been further refined to meet the special needs of atmospheric monitoring so that very small samples may now be efficiently tested. New in situ methods utilizing green plants actually avoid many of the usual difficulties of sample collection and preparation and offer special advantages in monitoring wastewater, sludges, and hazardous wastes. Insects also are being employed very effectively in the evaluation of gaseous air pollutants in controlled laboratory investigations. Increased emphasis has been placed on a comprehensive assessment of the potential of complex mixtures t9 cause various kinds of genetic damage. New as...

  1. Acute toxicity bioassay with native plants to evaluate an oil spill

    Directory of Open Access Journals (Sweden)

    Vivien Pentreath


    Full Text Available Plant bioassays are excellent tools for the evaluation of environmental risks. In particular the use of seeds of vascular plants is recommended due to their higher sensitivity. The aim of this study was to evaluate the behavior of native plants in order for them to be used as biological indicators of environmental oil pollution in relation to a standardized bioindicator. We analyzed the germination index (GI of Lactuca sativa L., Atriplex lampa (Moq. D.Dietri. and Prosopis denudans in thirty soil samples taken from an oil field to detect phytotoxic effects. GI, is a cheap, fast and reproducible biological method for determining the toxicity of the soil, thus helping to characterize areas with contaminated soils. The results show that, after germination, Atriplex lampa (Moq. D.Dietri. and Prosopis denudans are more resistant in the contaminated soils of an oil field than the reference bioindicator (Lactuca sativa L..

  2. A Bioassay Technique to Study Clomazone Residues in Sandy Loam Soil

    Directory of Open Access Journals (Sweden)

    Jelena Gajić Umiljendić


    Full Text Available A bioassay test was conducted to evaluate the sensitivity of maize, sunflower and barley toclomazone residues in sandy loam soil. Clomazone was applied at different rates from 0.12 to12 mg a.i./kg of soil. The parameters measured 14 days after treatment were: shoot height, freshand dry weight, and content of pigments (carotenoids, chlorophyll a and chlorophyll b. Theresults showed that the lowest clomazone concentration caused a significant reduction in allmeasured parameters for barley and sunflower shoots. Fresh weight of maize shoots was notsensitive to clomazone residual activity in soil while the other parameters were highly inhibited.Nomenclature: clomazone (2-(2-chlorbenzyl-4,4-dimethyl-1,2-oxazolidin-3-one, maize(Zea mays L., sunflower (Helianthus annuus L., barley (Hordeum vulgare L.

  3. Comparison of the results obtained by CALUX bioassay and GC-HRMS for different matrices

    Energy Technology Data Exchange (ETDEWEB)

    Carbonnelle, S.; Loco, J. van; Overmeire, I. van; Windal, I.; Wouwe, N. van; Goeyens, L. [Scientific Institute of Public Health, Brussels (Belgium); Cleuvenbergen, R. van [VITO, Mol (Belgium); Leeuwen, S. van [Netherlands Institute for Fisheries Research, Ijmuiden (Netherlands). Animal Science Group


    The reference method used to analyse polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) is chromatography with high resolution mass spectrometry (GC-HRMS). It is interesting to check the suitability of screening methods that are faster and less expensive. Different matrices (milk, fish oil, chicken compound feed, pork tissue, chicken tissue, sepiolitic clay, whole egg and herring tissue) were analysed in the frame of the European project DIFFERENCE1. One of the aims of this project is to optimise screening methods. The CALUX bio-assay was one of the screening techniques used. This paper presents the extraction and purification methods used for the analyses. The CALUX results for dioxins and for dl-PCBs were compared to the corresponding GC-HRMS results.

  4. Harvester ant bioassay for assessing hazardous chemical waste sites. [Pogonomyrmex owhyeei

    Energy Technology Data Exchange (ETDEWEB)

    Gano, K.A.; Carlile, D.W.; Rogers, L.E.


    A technique was developed for using harvester ants, Pogonomyrmex owhyeei, in terrestrial bioassays. Procedures were developed for maintaining stock populations, handling ants, and exposing ants to toxic materials. Relative toxicities were determined by exposing ants to 10 different materials. These materials included three insecticides, Endrin, Aldrin, and Dieldrin; one herbicide, 2,4-D; three complex industrial waste residuals, wood preservative sludge, drilling fluid, and slop oil; and three heavy metals, copper zinc, and cadium. Ants were exposed in petri dishes containing soil amended with a particular toxicant. Under these test conditions, ants showed no sensitivity to the metals or 2,4-D. Ants were sensitive to the insecticides and oils in repeated tests, and relative toxicity remained consistent throughout. Aldrin was the most toxic material followed by Dieldrin, Endrin, wood preservative sludge, drilling fluid, and slop oil. 12 refs., 2 figs., 2 tabs.

  5. Effects of injection speed of test samples on the mouse bioassay for paralytic shellfish poisoning toxins

    Directory of Open Access Journals (Sweden)

    Hodaka Suzuki


    Full Text Available The mouse bioassay has been used as the official method for paralytic shellfish poisoning toxins detection in Japan since 1980. However, differences in the results of this assay, when performed by different investigators, have been noted despite the use of the same sample. This study was performed to examine the effect of the injection speed, a hypothetical cause of such differences, on the death time of mice. Speed-controlled injection of the toxin (at 12, 6, 3, and 1.5 mL/min into mice was performed using a syringe pump, and the death times of mice were measured. No statistically significant differences were found among the groups, even between fast injection (5 s and very slow injection (40 s, indicating that the injection speed may not be the crucial factor for this assay.

  6. Robust Discrimination between Single Gold Nanoparticles and Their Dimers in Aqueous Solution for Ultrasensitive Homogeneous Bioassays

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi


    Full Text Available We propose a robust method to distinguish isolated single gold nanoparticles (AuNP monomers and their dimers under Brownian motion, a key for ultrasensitive homogeneous bioassays, including AuNP sandwich assays. To detect dimers and distinguish them from a larger number of monomers in aqueous solution, single-particle polarization microscopy was performed. For the accurate detection of individual particles, the optical anisotropy and rotational diffusion time are measured because a dimer is much more anisotropic than the nearly spherical monomer and the rotational diffusion time of a dimer is four times that of a monomer. By employing an autocorrelation analysis, we defined a measure of distinguishing that simultaneously enables high detection probability and low error probability. The detection platform offers homogeneous DNA hybridization assays and immunoassays at the subpicomolar level.

  7. Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen)

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Høj; Nielsen, Jesper Bo


    The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those...... of hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate...... solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over...

  8. Effects of Direct and Indirect Exposure of Insecticides to Garden Symphylan (Symphyla: Scutigerellidae) in Laboratory Bioassays. (United States)

    Joseph, Shimat V


    The garden symphylan, Scutigerella immaculata Newport, is a serious soil pest whose root feeding affects yield and survival of several high valued crops in the California's central coast. Because organophosphate insecticides, widely used for S. immaculata control, are rigorously regulated and little is known about the efficacy of alternate insecticides, laboratory bioassays were conducted to determine insecticide efficacy through repellency and lethality. To determine indirect repellency (noncontact) of insecticides, choice assays were conducted where five S. immaculata were introduced into the arena to choose between insecticide-treated and untreated wells whereas, in direct repellency (contact) assays, three insecticide-treated 1-cm-diameter discs were pasted into the arena and the number of visits, time spent per visitation, and number of long-duration (>10 s) stays of five S. immaculata were quantified. To determine efficacy through direct mortality, number of S. immaculata died after 72 h were determined by introducing 10 S. immaculata to insecticide-treated soil assays. In indirect exposure bioassays, seven (clothianidin, oxamyl, zeta-cypermethrin, chlorpyrifos, ethoprop, azadirachtin, and a combination of beta-cyfluthrin and imidacloprid) out of 14 insecticides tested elicited repellency to S. immaculata. Of six insecticides tested in the direct exposure assays, only tolfenpyrad elicited contact repellency. In soil assays, after 72 h of introduction, bifenthrin, oxamyl, clothianidin, zeta-cypermethrin, and tolfenpyrad caused 100, 95, 80, 44, and 44% S. immaculata mortality, respectively, which was significantly greater than distilled water and four other insecticides. The implications of these results on S. immaculata management in the California's central coast are discussed.

  9. A miniature bioassay for testing the acute phytotoxicity of photosystem II herbicides on seagrass. (United States)

    Wilkinson, Adam D; Collier, Catherine J; Flores, Florita; Mercurio, Phil; O'Brien, Jake; Ralph, Peter J; Negri, Andrew P


    Photosystem II (PSII) herbicides have been detected in nearshore tropical waters such as those of the Great Barrier Reef and may add to the pressure posed by runoff containing sediments and nutrients to threatened seagrass habitats. There is a growing number of studies into the potential effects of herbicides on seagrass, generally using large experimental setups with potted plants. Here we describe the successful development of an acute 12-well plate phytotoxicity assay for the PSII herbicide Diuron using isolated Halophila ovalis leaves. Fluorescence images demonstrated Diuron affected the entire leaf surface evenly and responses were not influenced by isolating leaves from the plant. The optimum exposure duration was 24 h, by which time the inhibition of effective quantum yield of PSII (∆F/F(m)') was highest and no deterioration of photosystems was evident in control leaves. The inhibition of ∆F/F(m)' by Diuron in isolated H. ovalis leaves was identical to both potted and hydroponically grown plants (with leaves remaining attached to rhizomes), indicating similar reductions in photosynthetic activity in these acute well-plate assays. The sensitivity of the assay was not influenced by irradiance (range tested 40 to 400 μmol photons m(-2) s(-1)). High irradiance, however, caused photo-oxidative stress in H. ovalis and this generally impacted in an additive or sub-additive way with Diuron to damage PSII. The bioassay using isolated leaves is more rapid, uses far less biological material and does not rely on specialised aquarium facilities in comparison with assays using potted plants. The development and validation of this sensitive bioassay will be useful to reliably screen and monitor the phytotoxicity of existing and emerging PSII herbicides and contribute to risk assessments and water quality guideline development in the future.

  10. Fate of soil-applied olive mill wastewater and potential phytotoxicity assessed by two bioassay methods. (United States)

    Saadi, Ibrahim; Raviv, Michael; Berkovich, Shimrit; Hanan, Aviva; Aviani, Ido; Laor, Yael


    Controlled land spreading of untreated olive mill wastewater (OMW) has been widely practiced as a means of its disposal. However, potential phytotoxic effects are critical for the selection of sites and crop types and for proper synchronization between land application and cropping. This study traced the fate of dissolved organic carbon (DOC), total phenols (TP), electrical conductivity, pH, microbial counts, and phytotoxicity to cress ( L.) after soil application at doses equivalent to 80, 160, and 320 m ha. Vertisol (fine-clayey) and Loess (sandy loam) soils were treated and incubated at 12 or 25°C and at moisture contents maintained at 70% of field water capacity or gradually reduced from 70 to 20% without compensation. Temperature, rather than moisture content, had a major effect on removal rates of DOC and TP. The maximum combined effect of warm temperature and higher moisture content resulted in removal rates greater than those under cooler, drier conditions by factors of up to 1.8 and 4.1 for DOC and TP, respectively. Favorable biodegradation conditions were indicated by increased numbers of total soil microorganisms and fungi by factors of up to 26 and 5, respectively. A whole-soil bioassay was developed to assess the dynamics of residual soil phytotoxicity after OMW application. Phytotoxicity measurement in soil extract generally showed stronger inhibition or stimulation activity than measurement in whole soil, depending on soil type and OMW dose. The newly developed bioassay seems to be useful for the refinement of general recommendations regarding permitted OMW application doses.

  11. A miniature bioassay for testing the acute phytotoxicity of photosystem II herbicides on seagrass.

    Directory of Open Access Journals (Sweden)

    Adam D Wilkinson

    Full Text Available Photosystem II (PSII herbicides have been detected in nearshore tropical waters such as those of the Great Barrier Reef and may add to the pressure posed by runoff containing sediments and nutrients to threatened seagrass habitats. There is a growing number of studies into the potential effects of herbicides on seagrass, generally using large experimental setups with potted plants. Here we describe the successful development of an acute 12-well plate phytotoxicity assay for the PSII herbicide Diuron using isolated Halophila ovalis leaves. Fluorescence images demonstrated Diuron affected the entire leaf surface evenly and responses were not influenced by isolating leaves from the plant. The optimum exposure duration was 24 h, by which time the inhibition of effective quantum yield of PSII (∆F/F(m' was highest and no deterioration of photosystems was evident in control leaves. The inhibition of ∆F/F(m' by Diuron in isolated H. ovalis leaves was identical to both potted and hydroponically grown plants (with leaves remaining attached to rhizomes, indicating similar reductions in photosynthetic activity in these acute well-plate assays. The sensitivity of the assay was not influenced by irradiance (range tested 40 to 400 μmol photons m(-2 s(-1. High irradiance, however, caused photo-oxidative stress in H. ovalis and this generally impacted in an additive or sub-additive way with Diuron to damage PSII. The bioassay using isolated leaves is more rapid, uses far less biological material and does not rely on specialised aquarium facilities in comparison with assays using potted plants. The development and validation of this sensitive bioassay will be useful to reliably screen and monitor the phytotoxicity of existing and emerging PSII herbicides and contribute to risk assessments and water quality guideline development in the future.

  12. Age-Related Differences in Susceptibility to Carcinogenesis: A Quantitative Analysis of Empirical Animal Bioassay Data (United States)

    Hattis, Dale; Goble, Robert; Russ, Abel; Chu, Margaret; Ericson, Jen


    In revising cancer risk assessment guidelines, the U.S. Environmental Protection Agency (EPA) analyzed animal cancer bioassay data over different periods of life. In this article, we report an improved analysis of these data (supplemented with some chemical carcinogenesis observations not included in the U.S. EPA’s original analysis) and animal bioassay studies of ionizing radiation. We use likelihood methods to avoid excluding cases where no tumors were observed in specific groups. We express dosage for animals of different weights on a metabolically consistent basis (concentration in air or food, or per unit body weight to the three-quarters power). Finally, we use a system of dummy variables to represent exposures during fetal, preweaning, and weaning–60-day postnatal periods, yielding separate estimates of relative sensitivity per day of dosing in these intervals. Central estimate results indicate a 5- to 60-fold increased carcinogenic sensitivity in the birth–weaning period per dose ÷ (body weight0.75-day) for mutagenic carcinogens and a somewhat smaller increase—centered about 5-fold—for radiation carcinogenesis per gray. Effects were greater in males than in females. We found a similar increased sensitivity in the fetal period for direct-acting nitrosoureas, but no such increased fetal sensitivity was detected for carcinogens requiring metabolic activation. For the birth–weaning period, we found an increased sensitivity for direct administration to the pups similar to that found for indirect exposure via lactation. Radiation experiments indicated that carcinogenic sensitivity is not constant through the “adult” period, but the dosage delivered in 12- to 21-month-old animals appears a few-fold less effective than the comparable dosage delivered in young adults (90–105 days of age). PMID:15289159

  13. Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay. (United States)

    Madsen-Bouterse, Sally A; Zhuang, Dongyue; O'Rourke, Katherine I; Schneider, David A


    The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.

  14. Evaluation of the toxic effects of arsenite, chromate, cadmium, and copper using a battery of four bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Ko, Kyung-Seok; Lee, Pyeong-Koo [Korea Institute of Geoscience and Mineral Resources (KIGAM), Daejeon (Korea, Republic of). Geologic Environment Div.; Kong, In Chul [Yeungnam Univ., Kyungbuk (Korea, Republic of). Dept. of Environmental Engineering


    The sensitivities of four different kinds of bioassays to the toxicities of arsenite, chromate, cadmium, and copper were compared. The different bioassays exhibited different sensitivities, i.e., they responded to different levels of toxicity of each of the different metals. However, with the exception of the {alpha}-glucosidase enzyme activity, arsenite was the most toxic compound towards all the tested organisms, exhibiting the highest toxic effect on the seeds of Lactuca, with an EC{sub 50} value of 0.63 mg/L. The sensitivities of Lactuca and Raphanus were greater than the sensitivities of two other kinds of seeds tested. Therefore, these were the seeds appropriate for use in a seed germination assay. A high revertant mutagenic ratio (5:1) of Salmonella typhimurium was observed with an arsenite concentration of 0.1 {mu}g/plate, indicative of a high possibility of mutagenicity. These different results suggested that a battery of bioassays, rather than one bioassay alone, is needed as a more accurate and better tool for the bioassessment of environmental pollutants. (orig.)

  15. Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products. (United States)

    Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Pinto, Terezinha de Jesus Andreoli; Lourenço, Felipe Rebello


    Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0μg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required.

  16. Long-Distance Signaling in bypass1 Mutants:Bioassay Development Reveals the bps Signal to Be a Metabolite

    Institute of Scientific and Technical Information of China (English)

    Emma Adhikari; Dong-Keun Lee; Patrick Giavalisco; Leslie E. Sieburth


    Root-to-shoot signaling is used by plants to coordinate shoot development with the conditions experienced by the roots.A mobile and biologically active compound,the bps signal,is over-produced in roots of an Arabidopsis thaliana mutant called bypass1 (bps1),and might also be a normally produced signaling molecule in wild-type plants.Our goal is to identify the bps signal chemically,which will then allow us to assess its production in normal plants.To identify any signaling molecule,a bioassay is required,and here we describe the development of a robust,simple,and quantitative bioassay for the bps signal.The developed bioassay follows the growth-reducing activity of the bps signal using the pCYCB1;1::GUS cell cycle marker.Wild-type plants carrying this marker,and provided the bps signal through either grafts or metabolite extracts,showed reduced cell division.By contrast,control grafts and treatment with control extracts showed no change in pCYCB1;1::GUS expression.To determine the chemical nature of the bps signal,extracts were treated with RNase A,Proteinase K,or heat.None of these treatments diminished the activity of bps1 extracts,suggesting that the active molecule might be a metabolite.This bioassay will be useful for future biochemical fractionation and analysis directed toward bps signal identification.

  17. Cumulative toxicity of an environmentally relevant mixture of nine regulated disinfection by-products in a multigenerational rat reproductive bioassay (United States)


  18. Studies of the Stimulus Specificity, Response Specificity, Process Specificity, and Task Specificity of the Behavioral Bioassay Phenomenon. Final Report. (United States)

    Braud, William G.

    If biochemical substrates and mechanisms could be identified, progress might be made in the detection and remediation of certain learning and memory disabilities. "Memory transfer" or "behavioral bioassay" methodology is a new technique developed for this purpose. It uses the behavior recipient animals to detect whatever chemicals are synthesized…

  19. A rational superfusion medium for the bioassay of E-type prostaglandins on the rat stomach strip

    NARCIS (Netherlands)

    Jager, L.P.; Hofman, G.A.; Noordwijk, J.V.


    The optimum composition of a mixture of antagonist to be used in the bioassay of E-type prostaglandins was determined for the rat stomach strip (RSS). In the presence of atropine (10t−7M), indomethacin (10t−6M), propranolol (10t−4M), and tolazoline (10t−4M) the sensitivity of the RSS to muscarinic,

  20. Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics. (United States)

    Finco, Deborah; Baltrukonis, Daniel; Clements-Egan, Adrienne; Delaria, Kathy; Gunn, George R; Lowe, John; Maia, Mauricio; Wong, Teresa


    Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference.

  1. A fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. (United States)

    Marty, Micah J; Pawlik, Joseph R


    Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

  2. Elemol and Amyris Oil Repel the Ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in Laboratory Bioassays (United States)

    The essential oil from Amyris balsamifera (Rutaceae) and elemol, a principal constituent of the essential oil of Osage orange, Maclura pomifera (Moraceae) were evaluated in in vitro and in vivo laboratory bioassays for repellent activity against host-seeking nymphs of the blacklegged tick, Ixodes sc...

  3. Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Hilscherova, K. [Dept. of Environmental Chemistry and Toxicology, Faculty of Science, Masaryk Univ., Brno (Czech Republic); Machala, M. [Veterinary Research Inst. of Veterinary Medicine, Brno (Czech Republic); Kannan, K.; Giesy, J.P. [Dept. of Zoology, National Food Safety and Toxicology Center, Inst. for Environmental Toxicology, Michigan State Univ., East Lansing, MI (United States); Blankenship, A.L. [Dept. of Zoology, National Food Safety and Toxicology Center, Inst. for Environmental Toxicology, Michigan State Univ., East Lansing, MI (United States); ENTRIX Inc., East Lansing, MI (United States)


    In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (AhR)- (hereafter referred as dioxin-like) or estrogen receptor (ER)-mediated activities of potential endocrine disrupting compounds (EDCs) in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQs) or estrogenic equivalents (E{sub 2}-EQs) and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Mass-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation. (orig.)

  4. Interpretation of bioassays in the study of interactions between soil organisms and plants: involvement of nutrient factors

    NARCIS (Netherlands)

    Troelstra, S.R.; Wagenaar, R.; Smant, W.; Peters, B.A.M.


    Increased plant growth in sterilized soil is usually ascribed to the elimination of (often unidentified) soil-borne pathogens. Plant-soil bioassays are reported here for three dune soils and two plant species (Ammophila arenaria and Carer arenaria). Dynamics of plant growth, availability and uptake

  5. Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1. (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J


    Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.

  6. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. (United States)

    Piva, Francesco; Ciaprini, Francesco; Onorati, Fulvio; Benedetti, Maura; Fattorini, Daniele; Ausili, Antonella; Regoli, Francesco


    Quality assessments are crucial to all activities related to removal and management of sediments. Following a multidisciplinary, weight of evidence approach, a new model is presented here for comprehensive assessment of hazards associated to polluted sediments. The lines of evidence considered were sediment chemistry, assessment of bioavailability, sub-lethal effects on biomarkers, and ecotoxicological bioassays. A conceptual and software-assisted model was developed with logical flow-charts elaborating results from each line of evidence on the basis of several chemical and biological parameters, normative guidelines or scientific evidence; the data are thus summarized into four specific synthetic indices, before their integration into an overall sediment hazard evaluation. This model was validated using European eels (Anguilla anguilla) as the bioindicator species, exposed under laboratory conditions to sediments from an industrial site, and caged under field conditions in two harbour areas. The concentrations of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons and trace metals were much higher in the industrial compared to harbour sediments, and accordingly the bioaccumulation in liver and gills of exposed eels showed marked differences between conditions seen. Among biomarkers, significant variations were observed for cytochrome P450-related responses, oxidative stress biomarkers, lysosomal stability and genotoxic effects; the overall elaboration of these data, as those of standard ecotoxicological bioassays with bacteria, algae and copepods, confirmed a higher level of biological hazard for industrial sediments. Based on comparisons with expert judgment, the model presented efficiently discriminates between the various conditions, both as individual modules and as an integrated final evaluation, and it appears to be a powerful tool to support more complex processes of environmental risk assessment.

  7. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds with both receptor types. (United States)

    Bovee, Toine F H; Helsdingen, Richard J R; Rietjens, Ivonne M C M; Keijer, Jaap; Hoogenboom, Ron L A P


    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hERalpha) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic compounds. Furthermore, a similar assay was developed based on the stable expression of human estrogen receptor beta (hERbeta). When exposed to 17beta-estradiol, the maximum transcriptional activity of the ERbeta cytosensor was only about 40% of the activity observed with ERalpha, but the concentration where half-maximal activation is reached (EC50), was about five times lower. The relative estrogenic potencies (REP), defined as the ratio between the EC50 of 17beta-estradiol and the EC50 of the compound, of the synthetic hormones dienestrol, hexestrol and especially mestranol were higher with ER, while DES was slightly more potent with ERbeta. The gestagens progesterone and medroxyprogesterone-acetate showed no response, whereas the androgen testosterone showed a very weak response. The anabolic agent, 19-nortestosterone showed a clear dose-related response with estrogen receptor but not beta. The phytoestrogens coumestrol, genistein, genistin, daidzein, daidzin and naringenin were relatively more potent with ERbeta. Ranking of the estrogenic potency with ER was: 17beta-estradiol > 8-prenylnaringenin > coumestrol > zearalenone > genistein > genistin > naringenin. The ranking with the ERbeta was: 17beta-estradiol > coumestrol > genistein > zearalenone > 8-prenylnaringen > daidzein > naringenin > genistin > daidzin. The hop estrogen 8-prenylnaringenin is relatively more potent with ERalpha. These data show that the newly developed bioassays are valuable tools for the rapid and high-throughput screening for estrogenic activity.

  8. Chip-Scale Magnetic Source of Cold Atoms (United States)


    30 3.7 The transistor motherboard and the 100 pin breakout board. . . . . . . . . . . . 31 3.8 The full structure of the slowing chip...a separate motherboard of 60 N-channel IRLB3813PbF International Rectifier MOSFET transistors. These transistors will be turned on and off by...60 wires from the slowing chip were soldered to a motherboard of 60 transistors, lined up in two rows of 30 on a 0.1 inch grid perforated prototyping

  9. Chip-scale Integrated Silica Ring Optical Gyro Project (United States)

    National Aeronautics and Space Administration — The next generation of space systems can benefit greatly by reduced c-SWaP integrated gyroscopes. By using photonic integration, it is possible to fit the entire...

  10. Chip-Scale Continuously Tunable Optical Orbital Angular Momentum Generator

    CERN Document Server

    Sun, Jie; Moresco, Michele; Coolbaugh, Douglas; Watts, Michael R


    Light carrying orbital angular momentum (OAM) has potential to impact a wide variety of applications ranging from optical communications to quantum information and optical forces for the excitation and manipulation of atoms, molecules, and micro-particles. The unique advantage of utilizing OAM in these applications relies, to a large extent, on the use of multiple different OAM states. Therefore, it is desirable to have a device that is able to gen- erate light with freely adjustable OAM states in an integrated form for large- scale integration. We propose and demonstrate a compact silicon photonic integrated circuit to generate a free-space optical beam with OAM state con- tinuously tuned from a single electrical input signal, realizing both integer and non-integer OAM states. The compactness and flexibility of the device and its compatibility with complementary metal-oxide-semiconductor (CMOS) pro- cessing hold promise for integration with other silicon photonic components for wide-ranging applications.

  11. A chip-scale integrated cavity-electro-optomechanics platform

    DEFF Research Database (Denmark)

    Winger, M.; Blasius, T. D.; Mayer Alegre, T. P.;


    We present an integrated optomechanical and electromechanical nanocavity, in which a common mechanical degree of freedom is coupled to an ultrahigh-Q photonic crystal defect cavity and an electrical circuit. The system allows for wide-range, fast electrical tuning of the optical nanocavity...... resonances, and for electrical control of optical radiation pressure back-action effects such as mechanical amplification (phonon lasing), cooling, and stiffening. These sort of integrated devices offer a new means to efficiently interconvert weak microwave and optical signals, and are expected to pave...

  12. Dynamical chaos in chip-scale optomechanical oscillators

    CERN Document Server

    Wu, Jiagui; Huang, Yongjun; Zhou, Hao; Yang, Jinghui; Liu, Jia-Ming; Yu, Mingbin; Lo, Guoqiang; Kwong, Dim-Lee; Xia, Guangqiong; Wong, Chee Wei


    Chaos has revolutionized the field of nonlinear science and stimulated foundational studies from neural networks, extreme event statistics, to physics of electron transport. Recent studies in cavity optomechanics provide a new platform to uncover quintessential architectures of chaos generation and the underlying physics. Here we report the first generation of dynamical chaos in silicon optomechanical oscillators, enabled by the strong and coupled nonlinearities of Drude electron-hole plasma. Deterministic chaotic oscillation is achieved, and statistical and entropic characterization quantifies the complexity of chaos. The correlation dimension D2 is determined at ~ 1.67 for the chaotic attractor, along with a maximal Lyapunov exponent rate about 2.94*the fundamental optomechanical oscillation. The corresponding nonlinear dynamical maps demonstrate the plethora of subharmonics, bifurcations, and stable regimes, along with distinct transitional routes into chaotic states. The chaos generation in our mesoscopic...

  13. Comparison and evaluation of a novel bioassay and high-performance liquid chromatography for the clinical measurement of serum voriconazole concentrations. (United States)

    Steinmann, Joerg; Huelsewede, Joerg; Buer, Jan; Rath, Peter-Michael


    The aim of this study was to develop and validate a novel bioassay for determining serum voriconazole (VRC) concentrations and to compare its routine clinical performance with that of high-performance liquid chromatography (HPLC). The biological activity of VRC was measured by a plate diffusion assay using a VRC-hypersusceptible Candida kefyr strain. The bioassay's utility was tested by measuring steady-state VRC concentrations in 100 serum probes from VRC-treated patients. The HPLC system used solvent extraction with hexane:dichloromethane followed by reversed-phase HPLC with ultraviolet detection. The intra-day and inter-day accuracy of the bioassay was bioassay (0.5 mg l(-1)). The result of linear regression analysis was HPLC = 1.0178 (bioassay) + 0.328; R(2) = 0.88; n = 100. Results of the serum panel ranged from 0.5 to more than 8.0 mg l(-1) for the bioassay and from 0.26 to 10.1 mg l(-1) for HPLC. Especially in laboratories without access to HPLC, the bioassay may be a clinically useful tool for therapeutic drug monitoring.

  14. Preliminary study on sediment bioassays with Mediterranean amphipod Corophium orientale; Studio preliminare per l`utilizzo dell`anfipode mediterraneo Corophium orientale nei saggi biologici su sedimenti marini

    Energy Technology Data Exchange (ETDEWEB)

    Bigongiari, N.; Giuliani, S. [CIBM, Centro Interuniversitario di Biologia Marina, Livorno (Italy); Pellegrini, D. [ICRAM, Istituto Centrale per la -ricerca Scientifica e Tecnologica Applicata al Mare, Rome (Italy); Meschini, P. [Acquario Comunale, Livorno (Italy)


    The amphipods are widely considered to be the most appropriate indicator organisms for sediment bioassay. Actually none Mediterranean species is considered for testing sediment by scientific literature. The aim of this work was to evaluate the fitness thresholds of Corophium orientale at different chemical-physical conditions (salinity and granulometry) and to propose this species in Mediterranean sediment bioassay. The results show a good sensitivity at the reference toxicant (Cd Cl{sub 2}) and an excellent adaptation at the different laboratory conditions. These preliminary results and the easy collection of the organisms make C. orientale a suitable organism for bioassay.

  15. Facile synthesis of methotrexate intercalated layered double hydroxides: Particle control, structure and bioassay explore

    Energy Technology Data Exchange (ETDEWEB)

    Tian, De-Ying; Liu, Zhen-Lei [Jiangsu Key Laboratory of Biofunctional Material, College of Chemistry and Material Science, Nanjing Normal University, Nanjing 210023 (China); Li, Shu-Ping, E-mail: [Jiangsu Key Laboratory of Biofunctional Material, College of Chemistry and Material Science, Nanjing Normal University, Nanjing 210023 (China); Li, Xiao-Dong [Jiangsu Key Laboratory of Biofunctional Material, College of Chemistry and Material Science, Nanjing Normal University, Nanjing 210023 (China); Shenzhen Research Institute of Xiamen University, Shenzhen 518057 (China)


    To study the influence of particle size on drug efficacy and other properties, a series of methotrexate intercalated layered double hydroxides (MTX/LDHs) were synthesized through the traditional coprecipitation method, using a mixture of water and polyethylene glycol (PEG-400) as the solvent. To adjust the particle size of MTX/LDHs, the dropping way, the volume ratio of water to PEG-400 and different hydrothermal treatment time changed accordingly, and the results indicate that the particle size can be controlled between 90 and 140 nm. Elemental C/H/N and inductive coupled plasma (ICP) analysis indicated that different synthesis conditions almost have no effect on the compositions of the nanohybrids. X-ray diffraction (XRD) patterns manifested the successful intercalation of MTX anions into the LDH interlayers, and it's also found out that different volume ratios of water to PEG-400 and variable dropping way can affect the crystallinity of the final samples, i.e., the volume ratio of 3:1 and pH decreasing are proved to be optimum conditions. Furthermore, both antiparallel monolayer and bilayers adopting different orientations are suggested for four samples from XRD results. Fourier transform infrared spectroscopy (FTIR) investigations proved the coexistence of CO{sub 3}{sup 2−} and MTX anions in the interlayer of the nanohybrids. MTX/LDH particles exhibited hexagonal platelet morphology with round corner and different dropping ways can affect the morphology greatly. Moreover, a DSC study indicated that longer time treatment can weaken the bond between the MTX anions and LDH layers. The kinetic release profiles told us that larger MTX/LDH particles have enhanced the ability of LDH layers to protect interlayer molecules. At last, the bioassay study indicated that the nanohybrids with larger diameters have higher tumor suppression efficiency. - Graphical abstract: A series of methotrexate intercalated layered double hydroxides were synthesized by changing the

  16. Comparative study on toxicity evaluation of anaerobically treated parboiled rice manufacturing wastewater through fish bioassay. (United States)

    Giri, Dipti Ramesh; Singh, Ekta; Satyanarayan, Shanta


    Short term aquatic bioassay has been developed into a useful tool in water quality management. These tests give information on comparative toxicity of several compounds. The objective of this study was to evaluate the acute toxicity of raw and anaerobically treated effluents of the parboiled rice manufacturing industry. The acute toxicity test was carried out by using the fish Lebistes reticulatus under laboratory conditions. LC50 values for 24, 48, 72 and 96 hours ranged between 4.6 and 7.0% for the raw parboiled rice manufacturing wastewater. Two anaerobic fixed film fixed bed reactors and two different media matrices, i.e. UV stabilized Biopac media and Fugino spirals, were used for the treatment of parboiled rice mill wastewater. Effluents from these two reactors depicted LC50 values in the range of 68-88% and 62-78% for Biopac and Fugino spiral packed reactors, respectively. From the results, it is evident that anaerobically treated effluents from Biopac packed reactor is marginally better than Fugino spiral packed reactor. Results subjected to statistical evaluation depicted regression coefficient of more than 0.9 indicating good correlation between the mortality and effluent concentration.

  17. Evaluation of coriander spice as a functional food by using in vitro bioassays. (United States)

    Zhang, Chuan-Rui; Dissanayake, Amila A; Kevseroğlu, Kudret; Nair, Muraleedharan G


    Coriander leaves and seeds are widely used as a condiment and spice. The use of roasted coriander seeds in food and beverage is very common. In this study, we investigated raw and roasted coriander seeds for their functional food quality using antioxidant, anti-inflammatory and human tumour cell proliferation inhibitory assays. The hexane and methanolic extracts of raw and roasted coriander seeds showed identical chromatographic and bioassay profiles. Chromatographic purification of the roasted seed extracts afforded tripetroselinin as the predominant component. Other isolates were petroselinic acid, 1,3-dipetroselinin, 2-C-methyl-d-erythritol, 2-C-methyl-d-erythritol 4-O-β-d-glucopyranoside and linalool. Hexane and methanolic extracts of both raw and roasted seeds and pure isolates from them showed comparable antioxidant and anti-inflammatory activities to the positive controls used in the assays, and inhibited the growth of human tumour cells AGS (gastric carcinoma), DU-145 and LNCaP (prostate carcinoma), HCT-116 (colon carcinoma), MCF-7 (breast carcinoma) and NCI-H460 (lung carcinoma) by 4-34%, respectively.

  18. Multiple linear and principal component regressions for modelling ecotoxicity bioassay response. (United States)

    Gomes, Ana I; Pires, José C M; Figueiredo, Sónia A; Boaventura, Rui A R


    The ecotoxicological response of the living organisms in an aquatic system depends on the physical, chemical and bacteriological variables, as well as the interactions between them. An important challenge to scientists is to understand the interaction and behaviour of factors involved in a multidimensional process such as the ecotoxicological response. With this aim, multiple linear regression (MLR) and principal component regression were applied to the ecotoxicity bioassay response of Chlorella vulgaris and Vibrio fischeri in water collected at seven sites of Leça river during five monitoring campaigns (February, May, June, August and September of 2006). The river water characterization included the analysis of 22 physicochemical and 3 microbiological parameters. The model that best fitted the data was MLR, which shows: (i) a negative correlation with dissolved organic carbon, zinc and manganese, and a positive one with turbidity and arsenic, regarding C. vulgaris toxic response; (ii) a negative correlation with conductivity and turbidity and a positive one with phosphorus, hardness, iron, mercury, arsenic and faecal coliforms, concerning V. fischeri toxic response. This integrated assessment may allow the evaluation of the effect of future pollution abatement measures over the water quality of Leça River.

  19. Assessment of toxic potential of Cerrado fruit seeds using Artemia salina bioassay

    Directory of Open Access Journals (Sweden)

    Raíza Cavalcante Fonseca


    Full Text Available Artemia salina bioassay was used to assess toxicity of seeds and kernels of Brazilian fruits from cerrado (central high plains region and other inner regions of the country. Water extracts of the kernels were filtered and added to Artemia cultures containing ten individuals per mL. Dose - response curves were constructed, and LD50 values were calculated. Pure potassium cyanide standard was used to draw a calibration curve for comparison to detect the presence of cyanide in the samples tested. Extracts of the seeds of araticum, mangaba, cagaita, jatobá, and tucumã were found toxic to Artemia salina, and some of the dose - response curves were very similar in shape to those obtained with pure potassium cyanide standards, while the samples of baru, cajá-manga, siriguela, trauma, and veludo showed no toxicity at all. The Guignard test, specific for cyanide detection, showed negative results in all toxic samples, suggesting the presence of other toxic compounds rather than cyanide. The comparison of araticum dose - response curve with those of other annonaceous fruits suggests the presence acetogenins as the main toxic compounds in the seeds. These results could be useful to prevent poisoning by industrial derivatives of the fruits studied.

  20. Ecotoxicological evaluation of polycyclic aromatic hydrocarbons using marine invertebrate embryo-larval bioassays. (United States)

    Bellas, Juan; Saco-Alvarez, Liliana; Nieto, Oscar; Beiras, Ricardo


    The toxicity of polycyclic aromatic hydrocarbons (PAHs) was determined using mussel, sea-urchin and ascidian embryo-larval bioassays. Fluorescent light exposure enhanced phenanthrene, fluoranthene, pyrene and hydroxypyrene toxicity in comparison with dark conditions, but not naphthalene and fluorene toxicity. The toxicity of PAHs was inversely related to their K(OW) values following QSAR models derived for baseline toxicity of general narcotics, whereas the obtained regression using toxicity data from photoactivated PAHs significantly departed from the general narcosis model. Also, the mixture toxicity of five PAHs to the larval growth of the sea-urchin was compared with predictions derived from the concentration addition concept, indicating less than additive effects. Finally, we compared our toxicity data with worst-case environmental concentrations in order to provide a preliminary estimate of the risk to the marine environment. Naphthalene, fluorene and pyrene are not considered to pose a risk to sea-urchin, mussel or ascidian larvae, whilst phenanthrene and fluoranthene may pose a risk for mussel and sea-urchin. Moreover, a higher risk for those species is expected when we consider the photoactivation of the PAHs.

  1. Productivity of chironomid larvae exposed to oil sands process water : in situ vs. lab bioassay results

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, K.; Ciborowski, J. [Windsor Univ., ON (Canada)


    Oil sands process water (OSPW) contains toxic concentrations of salts and napthenic acids that may compromise wetland reclamation efforts. The productivity of wetland biota is one of the criteria used by the Alberta government to determine if land leased to oil sands mining companies is restored. This study determined how chironomid productivity is influenced by the water from oil sands process material (OSPM) affected wetland. In this study, 26 10-day in situ and laboratory bioassays from water of three oil sands process material (OSPM) were compared with water from 3 reference wetlands to determine the influence of water from OSPM affected wetlands on chironomid productivity. Parallel studies were conducted with Chironomus riparius lab-cultured larvae and Chironomus sp larvae cultured from egg masses collected from an OSPW-affected wetland. In situ, chironomids were housed in small cylinders with fine-mesh netting to allow water exchange and contact with the sediment. Preliminary estimates of chironomids emerging from study wetlands indicated that native and lab cultured chironomids are not uniformly responsive to OSPW.

  2. Toxicological evaluation of landfill leachate using plant (Allium cepa) and fish (Leporinus obtusidens) bioassays. (United States)

    Klauck, Cláudia Regina; Rodrigues, Marco Antonio Siqueira; da Silva, Luciano Basso


    The disposal of municipal waste in landfills may pose an environmental problem because the product of the decomposition of these residues generates large volumes of leachate, which may present high toxicity. The aim of this study was to assess the toxic and genotoxic effects of a sample of untreated leachate in fish (Leporinus obtusidens) and onions (Allium cepa). The leachate was collected in a landfill located in the region of Vale do Rio dos Sinos, southern Brazil. The fish were exposed to raw leachate, at concentrations of 0.5%, 1.0%, 5%, 10% and 20% for 6 days, while the bulbs of A. cepa were exposed to concentrations of 5%, 10%, 25%, 50% and 100% for 48 h. For fish, the concentrations of 5%, 10% and 20% were lethal, thus indicating high toxicity; however, sublethal concentrations (0.5% and 1.0%) showed no genotoxicity by micronucleus test when compared with the control group. In the bioassays involving onions, high toxicity was observed, with significant reduction of root growth and mitotic index in bulbs exposed to the 100% concentration of the leachate. An increase in the frequency of chromosome abnormalities in the A. cepa root cells in anaphase-telophase was observed in accordance with the increase in the concentration of leachate (5%, 10%, 25% and 50%), with values significantly greater than the control, at the highest concentration. The results showed that the leachate contains toxic and genotoxic substances, thus representing a major source of environmental pollution if not handled properly.

  3. Results of an in-situ mussel bioassay in the Puget Sound

    Energy Technology Data Exchange (ETDEWEB)

    Houkal, D.; Rummel, B.; Shephard, B. [URS Consultants, Inc., Seattle, WA (United States)


    As part of an ecological evaluation in the Puget Sound, Washington, an in situ bioassay using the blue mussel (Mytilus galloprovincialis) was conducted to determine the effect of sediment-borne chemicals on bioaccumulation and growth of shellfish. The assay included four sample stations from a contaminated embayment (Sinclair Inlet) and one station from a reference site (Holmes Harbor). At each station, 300 mussels were deployed 1 meter above the sediment surface and maintained for a period of 3 months. The length and total weight of each mussel was measured at the beginning of the exposure period and the length, total weight, tissue weight, and shell weight of each mussel was measured at the end of the exposure period. Composite tissue samples from 100 mussels were collected at the beginning and end of the exposure period and analyzed for semivolatile organic chemicals, chlorinated pesticides, polychlorinated biphenyls, inorganic chemicals, organotin, and lipids. Water quality measurements (including temperature, salinity, dissolved oxygen, and chlorophyll a) were made at each station every two weeks during the assay to characterize environmental factors influencing mussel bioaccumulation and growth. Weight growth was similar among stations in Sinclair Inlet, but was significantly greater in all Sinclair Inlet stations compared to the Holmes Harbor reference station. Length growth was statistically indistinguishable among stations in Sinclair Inlet. Only one Sinclair Inlet station had a significantly greater length growth compared to the Holmes Harbor reference station. The influence of water quality on mussel growth is presented. The correlation between sediment chemistry and bioaccumulation is discussed.

  4. Acute toxicity bioassay of dimethoate on freshwater airbreathing catfish, Heteropneustes fossilis (Bloch). (United States)

    Pandey, Rakesh K; Singh, Ram N; Singh, Sarika; Singh, Narendra N; Das, Vijai K


    Pesticides are chemicals used for pest control in the agricultural fields. They finally reach the surrounding water bodies through surface runoff affecting the aquatic fauna. Dimethoate is frequently used organophosphate pesticide due to its high effectiveness and rapid breakdown into environmentally safe products. A 96 hr static acute toxicity test was carried out to determine the LC50 value of dimethoate, on the freshwater airbreathing catfish Heteropneustes fossilis (Bloch). The fish were exposed to 7 different concentrations of dimethoate (2.50, 2.75, 3.00, 3.25, 3.50, 3.75 and 4.00 mg l(-1)) for toxicity bioassay. Control (0.00 mg l(-1)) was also carried out. The data were subjected to Finney's Probit analysis and processed with Trimmed Spearman-Karber statistical software. The LC50 values for dimethoate for 24, 48, 72 and 96 hr were 3.38, 3.23, 3.08 and 2.98 mg l(-1), respectively. At higher concentration of dimethoate (3.25 mg l(-1) and above) the fish showed uncoordinated behaviour such as erratic and jerky swimming, attempt to jump out of water, frequent surfacing and gulping of air, decrease in opercular movement and copious secretion of mucus all over the body.

  5. Synthesis of nanostructured methotrexate/hydroxyapatite: Morphology control, growth mechanism, and bioassay explore. (United States)

    Dai, Chao-Fan; Li, Shu-Ping; Li, Xiao-Dong


    In this study, a new structure of methotrexate/hydroxyapatite (MTX/HAp) nanorods via a facile hydrothermal route was reported. The as-synthesized samples were then characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis. In order to explore the formation mechanism, the effects of reaction time, MTX concentrations and addition of ethylene glycol (PEG) were emphatically examined. The results indicated that, with the increase in reaction time, the fibrous nanoparticles turned to needle-like and then to rod-like. Our study also proved that reaction time of 12h was enough for the full-growth of the nanostructure. Drug-loading capacities (AIn) rose dramatically in the first 12h and reached a plateau afterwards. Importantly, MTX played a critical role in the longitudinal growth of MTX/HAp nanostructure and polyethylene glyco (PEG) was a good dispersing agent to improve the monodispersity. As expected, the functional agent of MTX was served as both the target anticancer drug loaded in HAp and effective complex agents to modify and control the morphologies of MTX/HAp. Lastly, in vitro bioassay tests gave us evidence that obvious tumor inhibition can be achieved when MTX was hybridized with HAp.

  6. Preparative Purification and Bioassay of Bt Toxin from Cry1Ab Transgenic Rice

    Institute of Scientific and Technical Information of China (English)

    WU Jian-min; YE Qin-fu


    A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa2CO3 and NaHCO3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L trisHCl buffer+1. 0 mmol/L EDTA, pH=8.0) mixed with 0. 1, 0. 3, 0. 5 and 0. 8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.

  7. Using the BioAssay Ontology for analyzing high-throughput screening data. (United States)

    Zander Balderud, Linda; Murray, David; Larsson, Niklas; Vempati, Uma; Schürer, Stephan C; Bjäreland, Marcus; Engkvist, Ola


    High-throughput screening (HTS) is the main starting point for hit identification in drug discovery programs. This has led to a rapid increase of available screening data both within pharmaceutical companies and the public domain. We have used the BioAssay Ontology (BAO) 2.0 for assay annotation within AstraZeneca to enable comparison with external HTS methods. The annotated assays have been analyzed to identify technology gaps, evaluate new methods, verify active hits, and compare compound activity between in-house and PubChem assays. As an example, the binding of a fluorescent ligand to formyl peptide receptor 1 (FPR1, involved in inflammation, for example) in an in-house HTS was measured by fluorescence intensity. In total, 155 active compounds were also tested in an external ligand binding flow cytometry assay, a method not used for in-house HTS detection. Twelve percent of the 155 compounds were found active in both assays. By the annotation of assay protocols using BAO terms, internal and external assays can easily be identified and method comparison facilitated. They can be used to evaluate the effectiveness of different assay methods, design appropriate confirmatory and counterassays, and analyze the activity of compounds for identification of technology artifacts.


    Directory of Open Access Journals (Sweden)

    Mohammad Kaisarul Islam


    Full Text Available Tinospora cordifolia (Fam: Menispermaceae has been investigated for evaluation of the biological activities. The stem of Tinospora cordifolia were extracted with carbon tetrachloride, n-hexane and methanol were collected and afford 4.0 mg, 4.0 mg, and 4.0 mg, respectively for the test. The crude carbon tetrachloride, n-hexane, and methanol extract of the plant were subjected to antimicrobial screening against 15 microorganisms such as gram-positive, gram-negative, fungi by the disc diffusion method. But interestingly no fraction showed any inhibitory effect against all the microorganisms. In the brine shrimp lethality bioassay, carbon tetrachloride fraction showed high toxicity, where LC90 value was only 6.25 mg/ml. The methanol crude extract and n-hexane fraction showed LC50 of 1.563 and 6.05 mg/ml respectively with very narrow therapeutic index (LC90 of 43.0 mg/ml and 45.0 mg/ml respectively. This indicated that the cytotoxicity exhibited by the carbon tetra chloride, n-hexane, and methanol extract was very significant.

  9. Bioassay and Identification of Root Exudates of Three Fruit Tree Species

    Institute of Scientific and Technical Information of China (English)

    Jiang-Hong Zhang; Zhi-Quan Mao; Li-Qin Wang; Huai-Rui Shu


    A laboratory bioassay was designed to determine the allelopathic potential of root exudates of three fruit tree species on apple germination. The results showed that root exudates of apple (Malus pumila L.) and peach (Prunus persica L.), each at concentrations of 0.02 and 0.2 mg/L, inhibited germination and radicle growth of apple seeds by 56.7%, 60.7%, 51.5%, and 59.3%, respectively. The corresponding shoot growth inhibition rate was 49.5%, 46.7%, 36.4%, and 44%, respectively. Root exudates of jujube (Ziziphus jujuba Mill.) had no significant effect on apple seeds.Qualitative determination of root exudates of apple, peach, and jujube tree was developed with gas chromatography-mass spectrometry. The root exudates of apple seedlings mainly contain organic acids, glycol, esters, and benzenphenol derivatives. Peach root exudates contained phenolic acids and benzenphenol derivatives in addition to two unidentified compounds. The root exudates of jujube did not contain any phenolic acids.

  10. Towards the development of an embryotoxicity bioassay with terrestrial snails: screening approach for cadmium and pesticides. (United States)

    Druart, Coline; Scheifler, Renaud; de Vaufleury, Annette


    Currently no bioassays are available to assess the embryotoxicity of chemicals with terrestrial soil invertebrates. We therefore presented a new method for embryotoxicity testing with snail eggs: a relevant biological material that incubates in soil and that can be exposed to contaminants from leachates and soil solution. The effects of aqueous solutions of two herbicide formulations, Reglone(®) (active ingredient (a.i.), diquat) and Roundup(®) or its a.i., glyphosate, of a surfactant (Agral(®) 90, a.i., nonylphenol polyethoxylates) and of cadmium (Cd) were studied. Endpoints were the hatching success and observations of embryo abnormalities after exposure. Roundup(®) was found to be more toxic than its a.i. alone (EC50(a.i.)=18 mg/l and about 1300 mg/l, respectively). Reglone(®) (EC50(a.i.)=0.72 mg/l) and Agral(®) (EC50(a.i.) ≈ 50 mg/l) were also tested together, revealing that Reglone(®) accounted for more than 99% of the mixture's toxicity. An antagonistic interaction between the two substances was found. For Cd (EC50=3.9 mg/l), a significant transfer from exposure medium to eggs was emphasized, particularly affecting the albumen. Abnormalities of embryogenesis in non-hatched embryos depended on the substance and the concentration considered.

  11. Reducing concentrations of ammonia to nontoxic levels prior to initiating whole-sediment bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Barrows, E.S.; Pinza, M.R.; Word, J.O. [Battelle/Marine Sciences Lab., Sequim, WA (United States); Greges, M.


    Determining the suitability of dredged material for ocean disposal requires whole-sediment toxicity tests with marine organisms. In addition to potential contaminants in dredged sediment, factors including grain size, salinity, hydrogen sulfide and ammonia can contribute to observed toxicity, thereby confounding test results. The presence of ammonia, a non-persistent but toxic compound, is particularly interesting because in open-water dredge and disposal situations, ammonia may not be a contaminant of concern. Sediment porewater ammonia concentrations in bioassays can be reduced to nontoxic levels before exposing organisms to permit evaluation of sediment contaminants without the additional toxicity effects of ammonia. Ten-day acute toxicity tests were conducted on sediments with Ampelisca abdita, Rhepoxynius abronius, Eohaustorius estuarius, and Mysidopsis bahia. Two procedures were followed to reduce ammonia prior to test initiation. In one, sediments in test chambers stood for 7 to 1 2 days while natural, nitrogen-reducing processes took place. In the other, sediments were tested in modified, flow-through systems. Ammonia was monitored in sediment porewater during ``purging`` periods, at test initiation and termination, and in overlying water during testing. Results were compared to the measured dose response of each species to ammonia, as determined by water-only reference toxicant tests. The question remains whether other sediment characteristics are affected by these procedures.

  12. Statistical evaluation of mortality in long-term carcinogenicity bioassays using a Williams-type procedure. (United States)

    Herberich, Esther; Hothorn, Ludwig A


    Several doses and a control group can be compared under order restriction using the Williams procedure for normally distributed endpoints assuming variance homogeneity. Comparison of the survival functions represents a secondary endpoint in long-term in vivo bioassays of carcinogenicity. Therefore, a Williams-type procedure for the comparison of survival functions is proposed for the assumption of the Cox proportional hazards model or the general frailty Cox model to allow a joint analysis over sex and strains. Interpretation according to both statistical significance and biological relevance is possible with simultaneous confidence intervals for hazard ratios. Related survival data can be analyzed using the R packages survival, coxme, and multcomp. Together with the R packages MCPAN and nparcomp, Dunnett- or Williams-type procedures are now available for the statistical analysis of the following endpoint types in toxicology: (i) normally distributed, (ii) non-normally distributed, (iii) score (ordered categorical) data, (iv) crude proportions, (v) survival functions, and (vi) time-to-tumor data with and without cause-of-death information.

  13. Development of a bioassay to assess the toxicity of oil sands sediments to pike (Esox lucius)

    Energy Technology Data Exchange (ETDEWEB)

    Turcotte, D.; Yuan, H.; Tumber, V.; Parrott, J. [Environment Canada, Ottawa, ON (Canada); Raine, J. [Saskatchewan Univ., Saskatoon, SK (Canada)


    Pike (Esox lucius) are a commercially sought fish species that inhabit the Athabasca River, which flows through the Athabasca oil sands. The fish are exposed to natural sources of bitumen from the McMurray formation. This study was conducted to design and implement a daily-renewal bioassay to assess the toxicity of oil sands to this fish species and to obtain information regarding the development of pike exposed to bitumen. Eggs were collected and fertilized with milt from spawning wild pike captured from Lake Diefenbaker in Saskatchewan. The fertilized eggs were exposed to different concentrations of sediments or culture water only (negative controls) until complete yolk absorption of control fish, approximately 15 days post-hatch. For the rest of the experiment, brine shrimp were fed to the walleye embryos every day after hatching. The developing fish were examined for morphological deformities, survival, hatching success, and changes in weight and length. The research findings indicated that pike is less sensitive than walleye and fathead minnow to the toxicity of oil sands sediments.

  14. Development of a bioassay using walleye (Sander vitreus) to assess the toxicity of oil sands sediments

    Energy Technology Data Exchange (ETDEWEB)

    Turcotte, D.; Yuan, H.; Tumber, V.; Parrott, J. [Environment Canada, Ottawa, ON (Canada); Raine, J. [Saskatchewan Univ., Saskatoon, SK (Canada)


    This study examined the effects of sediments from the Athabasca oil sands area on fish development and survival. Walleye (Sander vitreus) which inhabit the Athabasca River are exposed to natural sources of bitumen eroding from the McMurray formation. This study described the design and implementation of a daily-renewal bioassay to evaluate the potential effects of toxicants on walleye development. Eggs were collected and fertilized with milt from spawning wild walleye captured from Lake Diefenbaker in Saskatchewan. The fertilized eggs were exposed to different concentrations of sediments or culture water only (negative controls) until complete yolk absorption of control fish. The walleye embryos were fed brine shrimp daily after hatching and the developing fish were examined for morphological deformities, survival, hatching success, and changes in weight and length between treatments. Organics concentrations in fish tissues and water were measured when possible. Fathead minnows and northern pikes will also be exposed to the same sediments in order to compare the relative sensitivity of the three species.

  15. Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay.

    Directory of Open Access Journals (Sweden)

    Nicholas J Haley

    Full Text Available Chronic wasting disease (CWD is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose. The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD. Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice. In addition, PrP(CWD was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA. The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrP(CWD levels by traditional assays (western blot, ELISA and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP mice (373(+/-3 days in 2 of 9 urine-inoculated mice and 342(+/-109 days in 8 of 9 saliva-inoculated mice. These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

  16. Effects of Jatropha curcas oil in Lactuca sativa root tip bioassays. (United States)

    Andrade-Vieira, Larissa F; Botelho, Carolina M; Laviola, Bruno G; Palmieri, Marcel J; Praça-Fontes, Milene M


    Jatropha curcas L. (Euphorbiaceae) is important for biofuel production and as a feed ingredient for animal. However, the presence of phorbol esters in the oil and cake renders the seeds toxic. The toxicity of J. curcas oil is currently assessed by testing in animals, leading to their death. The identification of toxic and nontoxic improved varieties is important for the safe use of J. curcas seeds and byproducts to avoid their environmental toxicity. Hence, the aim of this study was to propose a short-term bioassay using a plant as a model to screen the toxicity of J. curcas oil without the need to sacrifice any animals. The toxicity of J. curcas oil was evident in germination, root elongation and chromosomal aberration tests in Lactuca sativa. It was demonstrated that J. curcas seeds contain natural compounds that exert phyto-, cyto- and genotoxic effects on lettuce, and that phorbol esters act as aneugenic agents, leading to the formation of sticky chromosomes and c-metaphase cells. In conclusion, the tests applied have shown reproducibility, which is important to verify the extent of detoxification and to determine toxic doses, thus reducing the numbers of animals that would be used for toxicity tests.

  17. Effects of Jatropha curcas oil in Lactuca sativa root tip bioassays

    Directory of Open Access Journals (Sweden)



    Full Text Available Jatropha curcas L. (Euphorbiaceae is important for biofuel production and as a feed ingredient for animal. However, the presence of phorbol esters in the oil and cake renders the seeds toxic. The toxicity of J. curcas oil is currently assessed by testing in animals, leading to their death. The identification of toxic and nontoxic improved varieties is important for the safe use of J. curcas seeds and byproducts to avoid their environmental toxicity. Hence, the aim of this study was to propose a short-term bioassay using a plant as a model to screen the toxicity of J. curcas oil without the need to sacrifice any animals. The toxicity of J. curcas oil was evident in germination, root elongation and chromosomal aberration tests in Lactuca sativa. It was demonstrated that J. curcas seeds contain natural compounds that exert phyto-, cyto- and genotoxic effects on lettuce, and that phorbol esters act as aneugenic agents, leading to the formation of sticky chromosomes and c-metaphase cells. In conclusion, the tests applied have shown reproducibility, which is important to verify the extent of detoxification and to determine toxic doses, thus reducing the numbers of animals that would be used for toxicity tests.

  18. Bioassay data and a retention-excretion model for systemic plutonium

    Energy Technology Data Exchange (ETDEWEB)

    Leggett, R.W.


    The estimation of systemic burdens from urinalyses has been the most common and useful method of quantifying occupational exposures to plutonium. Problems arise in using this technique, however, because of inadequate modeling of human retention, translocation, and excretion of this element. Present methods for estimating the systemic burden from urinalyses were derived to a large extent from patterns observed in the first few months after exposure, but there is now evidence that these same patterns do not persist over long periods. In this report we collect and discuss data needed for the interpretation of bioassay results for Pu. These data are used to develop a model that describes the movement, retention, and excretion of systemic Pu in the human body in terms of explicitly identified anatomical compartments. This model may be used in conjunction with existing models and/or case-specific information concerning the translocation of Pu from the respiratory or gastrointestinal tract or from wounds to the bloodstream. Attention is restricted to the behavior of Pu after it has gained access to the bloodstream. There remain significant uncertainties concerning some aspects of the movement of Pu, particularly its translocation from the liver. An attempt has been made to construct the model in such a way as to elucidate those areas needing further attention. 98 references, 18 figures, 16 tables.

  19. Double incision wound healing bioassay using Hamelia patens from El Salvador. (United States)

    Gomez-Beloz, Alfredo; Rucinski, James C; Balick, Michael J; Tipton, Camille


    Hamelia patens Jacq. (Rubiaceae) has received little attention in the laboratory for its wound healing ability even though it is commonly used as a treatment for wounds throughout Central America. A double incision wound healing bioassay was carried out with a crude extract of Hamelia patens collected from El Salvador. Animals were divided into three groups. Group I (n = 14) had the left incision treated with 5% (w/w) Hamelia patens and the contralateral side with petroleum jelly (PJ). Group II (n = 14) had the left incision treated with 10% (w/w) ointment and the contralateral side with petroleum jelly. Group III (n = 10) had the left incision treated with petroleum jelly and the contralateral side left untreated. Breaking strength of the incisions was measured on day 7 and day 12. For Groups I and II, there was no significant difference between treatment and control incisions at day 7. On day 12, there was a significant difference between the treated and control incisions for Groups I and II. There was no significant difference between petroleum jelly and untreated incisions for Group III on day 7 and day 12. Hamelia patens does increase breaking strength of wounds significantly more than the control group. Further wound healing studies of this plant are warranted.

  20. Bioassay-Directed Isolation of Active Compounds with Antiyeast Activity from a Cassia fistula Seed Extract

    Directory of Open Access Journals (Sweden)

    Subramanion L. Jothy


    Full Text Available Background and objective: Cassia fistula L belongs to the family Leguminosae, and it is one of the most popular herbal products in tropical countries. C. fistula seeds have been used as a herbal medicine and have pharmacological activity which includes anti-bacterial, anti-fungal, and antioxidant properties. The goal of this study was to identify compounds from C. fistula seeds which are responsible for anti-Candida albicans activity using bioassay-directed isolation. Results: The preliminary phytochemical screening of the plant seed revealed the presence of anthraquinones, flavonoids, saponins, tannins and terpenoids. The isolation of active compounds was carried out in four steps: multiple extractions, fractionation using column chromatography and purification using preparative thin-layer chromatography (TLC and liquid chromatography/mass spectrometry (LC/MS. The structure of separated compounds was determined on the basis of mass spectrometry data. One compound was identified is roseanone. Conclusions: The MS analysis on the active fraction from seed extract of C. fistula confirmed the presence of roseanone with antiyeast activity.

  1. Bed rest suppresses bioassayable growth hormone release in response to muscle activity (United States)

    McCall, G. E.; Goulet, C.; Grindeland, R. E.; Hodgson, J. A.; Bigbee, A. J.; Edgerton, V. R.


    Hormonal responses to muscle activity were studied in eight men before (-13 or -12 and -8 or -7 days), during (2 or 3, 8 or 9, and 13 or 14 days) and after (+2 or +3 and +10 or +11 days) 17 days of bed rest. Muscle activity consisted of a series of unilateral isometric plantar flexions, including 4 maximal voluntary contractions (MVCs), 48 contractions at 30% MVC, and 12 contractions at 80% MVC, all performed at a 4:1-s work-to-rest ratio. Blood was collected before and immediately after muscle activity to measure plasma growth hormone by radioimmunoassay (IGH) and by bioassay (BGH) of tibia epiphyseal cartilage growth in hypophysectomized rats. Plasma IGH was unchanged by muscle activity before, during, or after bed rest. Before bed rest, muscle activity increased (P muscle activity, a pattern that persisted through 8 or 9 days of bed rest. However, after 13 or 14 days of bed rest, plasma concentration of BGH was significantly lower after than before muscle activity (2,594 +/- 211 to 2,085 +/- 109 microg/l). After completion of bed rest, muscle activity increased BGH by 31% at 2 or 3 days (1,807 +/- 117 to 2,379 +/- 473 microg/l; P muscle activity.

  2. Skeletal muscle afferent regulation of bioassayable growth hormone in the rat pituitary (United States)

    Gosselink, K. L.; Grindeland, R. E.; Roy, R. R.; Zhong, H.; Bigbee, A. J.; Grossman, E. J.; Edgerton, V. R.


    There are forms of growth hormone (GH) in the plasma and pituitary of the rat and in the plasma of humans that are undetected by presently available immunoassays (iGH) but can be measured by bioassay (bGH). Although the regulation of iGH release is well documented, the mechanism(s) of bGH release is unclear. On the basis of changes in bGH and iGH secretion in rats that had been exposed to microgravity conditions, we hypothesized that neural afferents play a role in regulating the release of these hormones. To examine whether bGH secretion can be modulated by afferent input from skeletal muscle, the proximal or distal ends of severed hindlimb fast muscle nerves were stimulated ( approximately 2 times threshold) in anesthetized rats. Plasma bGH increased approximately 250%, and pituitary bGH decreased approximately 60% after proximal nerve trunk stimulation. The bGH response was independent of muscle mass or whether the muscles were flexors or extensors. Distal nerve stimulation had little or no effect on plasma or pituitary bGH. Plasma iGH concentrations were unchanged after proximal nerve stimulation. Although there may be multiple regulatory mechanisms of bGH, the present results demonstrate that the activation of low-threshold afferents from fast skeletal muscles can play a regulatory role in the release of bGH, but not iGH, from the pituitary in anesthetized rats.

  3. Lanthanide-containing polymer microspheres by multiple-stage dispersion polymerization for highly multiplexed bioassays. (United States)

    Abdelrahman, Ahmed I; Dai, Sheng; Thickett, Stuart C; Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Winnik, Mitchell A


    We describe the synthesis and characterization of metal-encoded polystyrene microspheres by multiple-stage dispersion polymerization with diameters on the order of 2 mum and a very narrow size distribution. Different lanthanides were loaded into these microspheres through the addition of a mixture of lanthanide salts (LnCl(3)) and excess acrylic acid (AA) or acetoacetylethyl methacrylate (AAEM) dissolved in ethanol to the reaction after about 10% conversion of styrene, that is, well after the particle nucleation stage was complete. Individual microspheres contain ca. 10(6)-10(8) chelated lanthanide ions, of either a single element or a mixture of elements. These microspheres were characterized one-by-one utilizing a novel mass cytometer with an inductively coupled plasma (ICP) ionization source and time-of-flight (TOF) mass spectrometry detection. Microspheres containing a range of different metals at different levels of concentration were synthesized to meet the requirements of binary encoding and enumeration encoding protocols. With four different metals at five levels of concentration, we could achieve a variability of 624, and the strategy we report should allow one to obtain much larger variability. To demonstrate the usefulness of element-encoded beads for highly multiplexed immunoassays, we carried out a proof-of-principle model bioassay involving conjugation of mouse IgG to the surface of La and Tm containing particles and its detection by an antimouse IgG bearing a metal-chelating polymer with Pr.

  4. Characterization of urinary volatiles in Swiss male mice (Mus musculus): bioassay of identified compounds

    Indian Academy of Sciences (India)

    S Achiraman; G Archunan


    The present study was carried out to investigate the chemical nature of the urine of male mice and to assess its bioactivity. Urine of mature male mice was extracted with dichloromethane (1 : 1 ratio v/v) and analysed by gas-chromatography linked mass-spectrometry (GC-MS). Ten different compounds such as alkanes, alcohols, etc. were detected in the urine. Among the ten, five compounds are specific to males, namely 3-cyclohexene-1-methanol (I), 3-amino-s-triazole (II), 4-ethyl phenol (III), 3-ethyl-2,7-dimethyl octane (IV) and 1-iodoundecane (V). The compound, 4-ethylphenol, has been previously reported in several strains of male mice. Furthermore, the compounds (II) and (IV) are similar to 2-sec-butylthiazole and dehydro-exo-brevicomin compounds which have already been reported in male mice. Bioassay revealed that compounds (II), (III) and (IV) were responsible for attracting females and in inducing aggression towards males, as compared to the other compounds, i.e. (I) and (V). The results indicate that these three volatiles (II, III and IV) of male mice appear to act as attractants of the opposite sex.

  5. Chronic toxicity bioassay with populations of the crustacean Artemia salina exposed to the organophosphate diazinon. (United States)

    Bustos-Obregon, Eduardo; Vargas, Alvaro


    A chronic toxicity bioassay was conducted with the microcrustacean Artemia salina as the testing organism for the toxic organophosphate diazinon in order to determine if the species is an appropriate indicator of pollution in aquatic environments. Tests of animal exposure to different concentrations of the toxicant were performed for 24, 48 and 72 hours after larvae hatching. Registered mortality data was used to obtain the lethal dose 50 (LD50) of the organophosphate for each exposure time, considering the immobilization of A. salina larvae as the mortality parameter. The lethal concentration (LD50) in the same exposure times was calculated by evaluating morphological changes on the three initial stages of larval development. Both doses were determined by using probit statistical analysis. Results indicate greater dose-response exactitude after 24 hours of exposure to the toxicant. High sensitivity of the organism to the toxicant was determined, thus indicating that A. salina is an appropriate ecotoxicological bioindicator of aquatic environments polluted with pesticides, with the special consideration that this species is a natural resident of saline water bodies, and thus could be used to control pollution in these environments as a result of the unrestrained usage of such toxic substances.

  6. Toxicity assessment of fumonisins using the brine shrimp (Artemia salina) bioassay. (United States)

    Hartl, M; Humpf, H U


    The Fusarium mycotoxins fumonisin B(1) (FB(1)) (1) and B(2) (FB(2)) (2), their hydrolysed analogues HFB(1) (3) and HFB(2) (4) and the recently discovered fumonisin derivatives N-palmitoyl-HFB(1) (5) and N-carboxymethyl-FB(1) (6) were compared for their toxicity in a short term bioassay using brine shrimp (Artemia salina). The brine shrimp were hatched in artificial sea water and exposed to the fumonisins in microwell plates with a mortality endpoint after 48 hours. LC(50) values were calculated after Probit transformation of the resulting data. Of the substances tested, fumonisin B(1) emerged to be the most toxic whereas its N-carboxymethyl analogue was 100-fold less effective. The hydrolysed fumonisins showed a four- to sixfold reduced toxicity compared to FB(1). N-Palmitoyl-HFB(1) had a higher LC(50) value than its precursor HFB(1). The brine shrimp assay proved to be a convenient and rapid system for toxicity assessment of this group of mycotoxins.

  7. Isolation of antibacterial compounds from Quercus dilatata L. through bioassay guided fractionation

    Directory of Open Access Journals (Sweden)

    Jamil Maryam


    Full Text Available Abstract Background Four medicinal plants (Chrozophora hierosolymitana Spreng, Chrysanthemum leucanthemum L., Ephedra gerardiana Wall. ex Stapf, and Quercus dilatata L. used by indigenous healers to treat various infectious diseases were selected for the present study. The major objective of the present study was isolation and characterization of antimicrobial components from the crude plant extracts using bioassay guided fractionation. Methods Seven methanolic extracts of the four plants were screened to identify any antimicrobial agents present in them. The active crude plant extract was fractionated first by solvent partitioning and then by HPLC. Characterization of the active fractions was done by using spectrophotometer. Results All the seven methanolic extracts showed low antifungal activity, however, when these extracts were tested for antibacterial activity, significant activity was exhibited by two extracts. The extract of aerial parts of Q. dilatata was most active and therefore, was selected for further analysis. Initially fractionation was done by solvent-solvent partitioning and out of six partitioned fractions, ethanol fraction was selected on the basis of results of antibacterial activity and phytochemical analysis. Further, fractionation was carried out by RP- HPLC and purified active subfractions were characterized by comparing their absorption spectra with that of the known natural products isolated from the plants of Quercus genus. Discussion and conclusion The results suggest that this is the first report of the isolated antibacterial compounds from this genus.

  8. Methotrexate intercalated layered double hydroxides with the mediation of surfactants: Mechanism exploration and bioassay study

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Chao-Fan; Tian, De-Ying; Li, Shu-Ping, E-mail:; Li, Xiao-Dong


    Methotrexatum intercalated layered double hydroxides (MTX/LDHs) hybrids were synthesized by the co-precipitation method and three kinds of nonionic surfactants with different hydrocarbon chain lengths were used. The resulting hybrids were then characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). XRD and FTIR investigations manifest the successful intercalation of MTX anions into the interlayer of LDHs. TEM graphs indicate that the morphology of the hybrids changes with the variation of the chain length of the surfactants, i.e., the particles synthesized using polyethylene glycol (PEG-7) present regular disc morphology with good monodispersity, while samples with the mediation of alkyl polyglycoside (APG-14) are heavily aggregated and samples with the addition of polyvinylpyrrolidone (PVP-10) exhibit irregular branches. Furthermore, the release and bioassay experiments show that monodisperse MTX/LDHs present good controlled-release and are more efficient in the suppression of the tumor cells. - Highlights: • Surfactants could be used to modify the dispersing state of MTX/LDHs hybrids. • Surfactants have great effect on the morphology of MTX/LDHs hybrids. • MTX/LDHs with good monodisperse degree are more efficient in the suppression of the tumor cells.

  9. Alkaloid presence and brine shrimp (Artemia salina) bioassay of medicinal species of eastern Nicaragua. (United States)

    Coe, Felix G; Parikh, Dimpi M; Johnson, Caley A


    We used an alkaloid test and a brine shrimp bioassay to assess the bioactivity of the medicinal plants used by eastern Nicaraguan healers in traditional medicine. Ethnomedicinal uses were obtained from interviews of traditional healers. Aqueous extracts derived from 30 species of angiosperms were assayed for the presence of alkaloids and toxicity. Species tested are distributed in 30 genera and 21 families. Of the 30 species tested for alkaloids with Dragendorff's reagent, 29 contained alkaloids. Toxicological analysis was conducted using the brine shrimp lethal assay (BSLA). Biological activity using BSLA was recorded as the median lethal concentration (LC50) that kills 50% of the larvae within 24 h of contact with the aqueous plant extracts. The LC50 of the shrimp was less than 2500 microg/mL for 3 (10%) species, 2500-5000 microg/mL for 9 (30%), 5001-7500 microg/mL for 7 (23%), 7501-10000 microg/mL for 3 (10%), and greater than 10000 microg/mL for 8 (27%) of the species. The members of the orders Santales and Rubiales in general contained species with greater toxicity than any other group. Struthanthus cassythoides (Struthanthus cassythoides Millsp.(Loranthaceae)). (LC50 1574 microg/mL) and Alibertia edulis (Rich.) A. Rich. (Rubiaceae) (LC50 1741 microg/mL) were the most toxic.

  10. Towards the development of an embryotoxicity bioassay with terrestrial snails: Screening approach for cadmium and pesticides

    Energy Technology Data Exchange (ETDEWEB)

    Druart, Coline; Scheifler, Renaud [Department of Chrono-Environment, University of Franche-Comte, UMR UFC/CNRS 6249 usc INRA, Place Leclerc, F-25030 Besancon Cedex (France); Vaufleury, Annette de, E-mail: [Department of Chrono-Environment, University of Franche-Comte, UMR UFC/CNRS 6249 usc INRA, Place Leclerc, F-25030 Besancon Cedex (France)


    Currently no bioassays are available to assess the embryotoxicity of chemicals with terrestrial soil invertebrates. We therefore presented a new method for embryotoxicity testing with snail eggs: a relevant biological material that incubates in soil and that can be exposed to contaminants from leachates and soil solution. The effects of aqueous solutions of two herbicide formulations, Reglone (active ingredient (a.i.), diquat) and Roundup or its a.i., glyphosate, of a surfactant (Agral 90, a.i., nonylphenol polyethoxylates) and of cadmium (Cd) were studied. Endpoints were the hatching success and observations of embryo abnormalities after exposure. Roundup was found to be more toxic than its a.i. alone (EC50{sub a.i.} = 18 mg/l and about 1300 mg/l, respectively). Reglone (EC50{sub a.i.} = 0.72 mg/l) and Agral (EC50{sub a.i.} {approx} 50 mg/l) were also tested together, revealing that Reglone accounted for more than 99% of the mixture's toxicity. An antagonistic interaction between the two substances was found. For Cd (EC50 = 3.9 mg/l), a significant transfer from exposure medium to eggs was emphasized, particularly affecting the albumen. Abnormalities of embryogenesis in non-hatched embryos depended on the substance and the concentration considered.

  11. A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins. (United States)

    Turcotte, Carl; Lacroix, Christophe; Kheadr, Ehab; Grignon, Louis; Fliss, Ismaïl


    A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r(2)=0.992+/-0.004) was obtained for the exponential regression in the nisin Z concentration range of 20-120 ng/ml with 1 x 10(7) CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r(2)=0.993+/-0.005, a precision (8.2%) and an accuracy lower than 15%.

  12. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists. (United States)

    Bovee, Toine F H; Helsdingen, Richard J R; Hamers, Astrid R M; van Duursen, Majorie B M; Nielen, Michel W F; Hoogenboom, Ron L A P


    Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC(50) of 17beta-testosterone and the EC(50) of the compound, of 5alpha-dihydrotestosterone, methyltrienolone, and 17beta-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

  13. General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay. (United States)

    Mallardi, Antonia; Angarano, Valeria; Magliulo, Maria; Torsi, Luisa; Palazzo, Gerardo


    A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 μM, respectively, have been obtained.

  14. Using a macroalgal δ15N bioassay to detect cruise ship waste water effluent inputs. (United States)

    Kaldy, James


    Green macroalgae bioassays were used to determine if the δ15N signature of cruise ship waste water effluent (CSWWE) could be detected in a small harbor. Opportunistic green macroalgae (Ulva spp.) were collected, cultured under nutrient depleted conditions and characterized with regard to N content and δ15N. Samples of algae were used in controlled incubations to evaluate the direction of isotope shift from exposure to CSWWE. Algae samples exposed to CSWWE exhibited an increase of 1-2.5‰ in δ15N values indicating that the CSWWE had an enriched isotope signature. In contrast, algae samples exposed to field conditions exhibited a significant decrease in the observed δ15N indicating that a light N source was used. Isotopically light, riverine nitrogen derived from N2-fixing trees in the watershed may be a N source utilized by algae. These experiments indicate that the δ15N CSWWE signature was not detectable under the CSWWE loading conditions of this experiment.

  15. Strategies of reducing the carcinogenic risk of cytostatic agents on the basis of bioassay evaluation. (United States)

    Berger, M R


    This article described strategies that can be used to reduce the carcinogenic risk of cytostatic chemotherapy and summarizes our recent experimental results. Reduction of neoplasms caused by the carcinogenic potency inherent in cytostatic agents can be obtained. (A) by chemical modifications such as: (1) exchanging a chlorine atom in N, N'-bis-(2-chloroethyl)-N-nitrosourea (BCNU) in the chloroethyl group at N'-position for a hydroxyl group to form the less carcinogenic analog N-(2-chloroethyl)-N'-(2-hydroxyethyl)-N-nitrosourea (HECNU); (2) linking chlorambucil to the steroid prednisolone to obtain a conjugate (prednimustine) with distinctly lower carcinogenic potential than chlorambucil; (3) progressive ring halogenation of phenyl-triazenes to generate agents with decreased long-term toxic risk; (B) by replacing cyclophosphamide within the carcinogenic drug combination of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) by vincristine to form the combination VMF which has no detectable carcinogenic potential; (C) by coadministration of cyclophosphamide and mesna to achieve a dose-related reduction of cyclophosphamide-induced urinary bladder carcinomas; (D) by administration of dinaline, a compound which reduces the spontaneous incidence of malignant tumors in rats. These examples demonstrate that the carcinogenic risk of single agents and drug combinations used for antineoplastic chemotherapy has successfully been reduced, as assessed in long-term bioassays. Such strategies should be considered in the treatment of patients with long life expectancy following cytotoxic chemotherapy.

  16. Phytochemical and toxicity evaluation ofPhaleria macrocarpa (Scheff.) Boerl by MCF-7 cell line and brine shrimp lethality bioassay

    Institute of Scientific and Technical Information of China (English)

    Abul Kalam Azad; Wan Mohd Azizi Wan Sulaiman; Nushrat Khan Sunzida


    Objective:To evaluate the cytotoxicity ofPhaleria macrocarpa fruits extracts. Methods: The cytotoxicity test was carried out byin vitroMCF-7 cell line andin vivo brine shrimp lethality bioassay. Results: The preliminary phytochemical test showed the presence of alkaloids, carbohydrate, glycosides, saponin, terpene, steroids, phenols and flavonoids. TheMTT-assay results showed that the highest percentage of cell viability was 106.23% at concentration of 1.25µL and the lowest percentage was 13.04% at concentration of 10µL. Conclusions:TheMTT-assay and brine shrimp lethality bioassay results showed that the extract was non-toxic and it would be consumable as a herbal remedy.

  17. Bioassay evaluation on the efficacy of α-cypermethrin impregnated into long lasting insecticide treated nets using Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    Vatandoost Hassan; Mamivand Poor Hossein; Shayeghi Mansoreh; Abai Mohamad Reza; Raeisi Ahmad; Nikpoor Fatemeh


    Objective: To evaluate the bioefficacy of α-cypermethrin impregnated into long lasting insecticide treated nets (LLITNs- INTERCEPTOR®) against main malaria vector, Anopheles stephensi (An. stephensi). Methods: The effectiveness of bed net impregnated withα-cypermethrin (INTERCEPTOR®) with washing was evaluated. The washing procedure and bioassay tests were carried out according to the WHO-recommended methods. Malaria vector, An. stephensi was exposed to impregnated bed net for three minutes and then mortality measured after 24 h recovery period. Knockdown was also measured according to the logarithmic times. Results:Result of cone bioassay method showed that bioefficacy ofα-cypermethrin decreased from 100%in unwashed to 15%in 20 washes. KT50 was measure as one minute in one wash and increased to 40 min in 20 washes. Discussion: Findings of this study provide guideline for malaria vector control authorities and people using pyrethroid-impregnated bed nets.

  18. Multiple Applications of Alamar Blue as an Indicator of Metabolic Function and Cellular Health in Cell Viability Bioassays

    Directory of Open Access Journals (Sweden)

    Sephra N. Rampersad


    Full Text Available Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls.

  19. Cascade bioassay evidence for the existence of urothelium-derived inhibitory factor in Guinea pig urinary bladder. (United States)

    Guan, Na N; Thor, Anna; Hallén, Katarina; Wiklund, N Peter; Gustafsson, Lars E


    Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1-5 µM in the presence of scopolamine 5-30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.

  20. Immunoassay and Nb2 lymphoma bioassay prolactin levels and mammographic density in premenopausal and postmenopausal women the Nurses' Health Studies. (United States)

    Rice, Megan S; Tworoger, Shelley S; Bertrand, Kimberly A; Hankinson, Susan E; Rosner, Bernard A; Feeney, Yvonne B; Clevenger, Charles V; Tamimi, Rulla M


    Higher circulating prolactin levels have been associated with higher percent mammographic density among postmenopausal women in some, but not all studies. However, few studies have examined associations with dense area and non-dense breast area breast or considered associations with prolactin Nb2 lymphoma cell bioassay levels. We conducted a cross-sectional study among 1,124 premenopausal and 890 postmenopausal women who were controls in breast cancer case-control studies nested in the Nurses' Health Study (NHS) and NHSII. Participants provided blood samples in 1989-1990 (NHS) or 1996-1999 (NHSII) and mammograms were obtained from around the time of blood draw. Multivariable linear models were used to assess the associations between prolactin levels (measured by immunoassay or bioassay) with percent density, dense area, and non-dense area. Among 1,124 premenopausal women, percent density, dense area, and non-dense area were not associated with prolactin immunoassay levels in multivariable models (p trends = 0.10, 0.18, and 0.69, respectively). Among 890 postmenopausal women, those with prolactin immunoassay levels in the highest versus lowest quartile had modestly, though significantly, higher percent density (difference = 3.01 percentage points, 95 % CI 0.22, 5.80) as well as lower non-dense area (p trend = 0.02). Among women with both immunoassay and bioassay levels, there were no consistent differences in the associations with percent density between bioassay and immunoassay levels. Postmenopausal women with prolactin immunoassay levels in the highest quartile had significantly higher percent density as well as lower non-dense area compared to those in the lowest quartile. Future studies should examine the underlying biologic mechanisms, particularly for non-dense area.

  1. Strain typing of classical scrapie by transgenic mouse bioassay using protein misfolding cyclic amplification to replace primary passage.

    Directory of Open Access Journals (Sweden)

    Katy E Beck

    Full Text Available According to traditional murine bioassay methodology, prions must be serially passaged within a new host before a stable phenotype, and therefore a strain, can be assigned. Prions often transmit with difficulty from one species to another; a property termed the transmission barrier. Transgenic mouse lines that over express prion protein (PrP genes of different species can circumvent the transmission barrier but serial passages may still be required, particularly if unknown strains are encountered. Here we sought to investigate whether protein misfolding cyclic amplification (PMCA, an in-vitro method of PrP(Sc replication, could be used to replace serial passage of VRQ/VRQ classical scrapie isolates undergoing strain typing in ovine transgenic tg338 mice. Two classical scrapie field isolates that do not readily transmit to wild-type mice underwent bioassay in tg338 mice pre- and post- PMCA and the phenotype of disease in inoculated mice was compared. For one of the sources investigated, the PMCA product gave rise to the same disease phenotypes in tg338 mice as traditional bioassay, as indicated by lesion profile, IHC analysis and Western blot, whilst the second source produced phenotypic characteristics which were not identical with those that arose through traditional bioassay. These data show that differences in the efficiency of PMCA as a strain-typing tool may vary between ovine classical scrapie isolates and therefore suggest that the ability of PMCA to replace serial passage of classical scrapie in tg338 mice may depend on the strain present in the initial source.

  2. Bioassays with caged hyalella azteca to determine in situ toxicity downstream of two Saskatchewan, Canada, uranium operations. (United States)

    Robertson, Erin L; Liber, Karsten


    The main objectives of this in situ study were to evaluate the usefulness of an in situ bioassay to determine if downstream water bodies at the Key Lake and Rabbit Lake uranium operations (Saskatchewan, Canada) were toxic to Hyalella azteca and, if toxicity was observed, to differentiate between the contribution of surface water and sediment contamination to in situ toxicity. These objectives were achieved by performing 4-d in situ bioassays with laboratory-reared H. azteca confined in specially designed, paired, surface water and sediment exposure chambers. Results from the in situ bioassays revealed significant mortality, relative to the respective reference site, at the exposure sites at both Key Lake (p azteca at both operations, although this relationship was stronger at Key Lake. At Key Lake, the primary cause of aquatic toxicity to H. azteca did not appear to be correlated with the variables measured in this study, but most likely with a pulse of organic mill-process chemicals released during the time of the in situ study-a transient event that was caused by a problem with the mill's solvent extraction process. The suspected cause of in situ toxicity to H. azteca at Rabbit Lake was high levels of uranium in surface water, sediment, and pore water.

  3. Use of plant and earthworm bioassays to evaluate remediation of soil from a site contaminated with polychlorinated biphenyls

    Energy Technology Data Exchange (ETDEWEB)

    Meier, J.R.; Chang, L.W.; Meckes, M.C.; Smith, M.K. [Environmental Protection Agency, Cincinnati, OH (United States); Jacobs, S. [DynCorp, Cincinnati, OH (United States); Torsella, J. [Oak Ridge Inst. of Science and Education, Cincinnati, OH (United States)


    Soil from a site heavily contaminated with polychlorinated biphenyls (PCBs) was treated with a pilot-scale, solvent extraction technology. Bioassays in earthworms and plants were used to examine the efficacy of the remediation process for reducing the toxicity of the soil. The earthworm toxicity bioassays were the 14-d survival test and 21-d reproduction test, using Lumbricus terrestris and Eisenia fetida andrei. The plant bioassays included phytotoxicity tests for seed germination and root elongation in lettuce and oats, and a genotoxicity test (anaphase aberrations) in Allium cepa (common onion). Although the PCB content of the soil was reduced by 99% (below the remediation goal), toxicity to earthworm reproduction remained essentially unchanged following remediation. Furthermore, phytotoxicity and genotoxicity were higher for the remediated soil compared to the untreated soil. The toxicity remaining after treatment appeared to be due to residual solvent introduced during the remediation process, and/or to heavy metals or other inorganic contaminants not removed by the treatment. Mixture studies involving isopropanol and known toxicants indicated possible synergistic effects of the extraction solvent and soil contaminants. The toxicity in plants was essentially eliminated by a postremediation, water-rinsing step. These results demonstrate a need for including toxicity measurements in the evaluation of technologies used in hazardous waste site remediations, and illustrate the potential value of such measurements for making modifications to remediation processes.

  4. Identification of antifungal natural products via Saccharomyces cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and mode of action. (United States)

    Tebbets, Brad; Yu, Zhiguo; Stewart, Douglas; Zhao, Li-Xing; Jiang, Yi; Xu, Li-Hua; Andes, David; Shen, Ben; Klein, Bruce


    Since current antifungal drugs have not kept pace with the escalating medical demands of fungal infections, new, effective medications are required. However, antifungal drug discovery is hindered by the evolutionary similarity of mammalian and fungal cells, which results in fungal drug targets having human homologs and drug non-selectivity. The group III hybrid histidine kinases (HHKs) are an attractive drug target since they are conserved in fungi and absent in mammals. We used a Saccharomyces cerevisiae reporter strain that conditionally expresses HHK to establish a high-throughput bioassay to screen microbial extracts natural products for antifungals. We identified macrotetrolides, a group of related ionophores thought to exhibit restricted antifungal activity. In addition to confirming the use of this bioassay for the discovery of antifungal natural products, we demonstrated broader, more potent fungistatic activity of the macrotetrolides against multiple Candida spp., Cryptococcus spp., and Candida albicans in biofilms. Macrotetrolides were also active in an animal model of C. albicans biofilm, but were found to have inconsistent activity against fluconazole-resistant C. albicans, with most isolates resistant to this natural product. The macrotetrolides do not directly target HHKs, but their selective activity against S. cerevisiae grown in galactose (regardless of Drk1 expression) revealed potential new insight into the role of ion transport in the mode of action of these promising antifungal compounds. Thus, this simple, high-throughput bioassay permitted us to screen microbial extracts, identify natural products as antifungal drugs, and expand our understanding of the activity of macrotetrolides.

  5. Assessment of the ecotoxicological risk of combined sewer overflows for an aquatic system using a coupled "substance and bioassay" approach. (United States)

    Gooré Bi, Eustache; Monette, Frederic; Gasperi, Johnny; Perrodin, Yves


    Very few tools are available for assessing the impact of combined sewer overflows (CSOs) on receiving aquatic environments. The main goal of the study was to assess the ecotoxicological risk of CSOs for a surface aquatic ecosystem using a coupled "substance and bioassay" approach. Wastewater samples from the city of Longueuil, Canada CSO were collected for various rainfall events during one summer season and analyzed for a large panel of substances (n = 116). Four bioassays were also conducted on representative organisms of surface aquatic systems (Pimephales promelas, Ceriodaphnia dubia, Daphnia magna, and Oncorhynchus mykiss). The analytical data did not reveal any ecotoxicological risk for St. Lawrence River organisms, mainly due to strong effluent dilution. However, the substance approach showed that, because of their contribution to the ecotoxicological hazard posed by the effluent, total phosphorus (Ptot), aluminum (Al), total residual chlorine, chromium (Cr), copper (Cu), pyrene, ammonia (N-NH4 (+)), lead (Pb), and zinc (Zn) require more targeted monitoring. While chronic ecotoxicity tests revealed a potential impact of CSO discharges on P. promelas and C. dubia, acute toxicity tests did not show any effect on D. magna or O. mykiss, thus underscoring the importance of chronic toxicity tests as part of efforts aimed at characterizing effluent toxicity. Ultimately, the study leads to the conclusion that the coupled "substance and bioassay" approach is a reliable and robust method for assessing the ecotoxicological risk associated with complex discharges such as CSOs.

  6. Detection of estrogen receptor endocrine disruptor potency of commonly used organochlorine pesticides using the LUMI-CELL ER bioassay

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, J.D.; Chu, A.C.; Clark, G.C. [Xenobiotic Detection Systems, Inc., Durham, NC (United States); Chu, M.D. [Alta Analytical Perspectives, Wilmington, NC (United States); Denison, M.S. [Dept. of Environmental Toxicology, Univ. of California, Davis, CA (United States)


    In order to detect the endocrine disrupting potency of organochlorine pesticides and other compounds, BG-1 (human ovarian carcinoma) cells containing a stably transfected estrogenresponsive luciferase reporter gene plasmid (BG1Luc4E2), was used. This cell line, termed the LUMI-CELL trademark ER estrogenic cell bioassay system, responds in a time-, dose dependent- and chemical-specific manner with the induction of luciferase gene expression in response to exposure to estrogen (but not other steroid hormones) and estrogenic chemicals in a high-throughput screening (HTPS) format6. Here we describe studies in which the LUMI-CELL trademark ER estrogenic cell bioassay system was used for high throughput screening (HTPS) analysis of the estrogenic disrupting potency of several commonly used pesticides and organochlorines: p,p'DDT; p,p'-DDE; DDD; {alpha}a-chlordane; {psi}-chlordane; Kepone; Methoxychlor; Vinclozolin; Fenarimol; 2,4,5-Trichlorophenoxyacetic Acid; and Dieldrin. Our results demonstrate the utility of XDS's LUMI-CELL trademark ER bioassay HTPS system for screening chemicals for estrogenic activity.

  7. Biological testing of sediment for the Olympia Harbor Navigation Improvement Project, 1988: Geoduck, amphipod, and echinoderm bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Ward, J.A.; Word, J.Q.; Antrim, L.D.


    The Olympia Harbor Navigation Improvement Project requires the dredging of approximately 330,000 cubic yards (cy) of sediment from the harbor entrance channel and 205,185 cy from the turning basin. Puget Sound Dredged Disposal Analysis (PSDDA) partial characterization studies were used to plan a full sediment characterization in which chemical analyses and biological testing of sediments evaluated the suitability of the dredged material for unconfined, open-water disposal. The US Army Corps of Engineers (COE), Seattle District, contracted with NOAA/NMFS, Environmental Conservation Division, to perform the chemical analysis and Microtox bioassay tests, and with the Battelle/Marine Sciences Laboratory (MSL) in Sequim to perform flow-through solid-phase bioassays utilizing juvenile (8 to 10 mm) geoduck clams, Panopea generosa, and static solid phase bioassays using the phoxocephalid amphipod, Rhepoxynius abronius, developing embryos and gametes of the purple sea urchin, Strongylocentrotus purpuratus, and the larvae of the Pacific oyster Crassostrea gigas. When the results of the biological tests were evaluated under PSDDA guidelines, it was found that all the tested sediment treatments from Olympia Harbor are suitable for unconfined open-water disposal. 14 refs., 12 figs., 3 tabs.

  8. Assessment of field-related influences on polychlorinated biphenyl exposures and sorbent amendment using polychaete bioassays and passive sampler measurements (United States)

    Janssen, E.M.; Oen, A.M.; Luoma, S.N.; Luthy, R.G.


    Field-related influences on polychlorinated biphenyl (PCB) exposure were evaluated by employing caged deposit-feeders, Neanthes arenaceodentata, along with polyoxymethylene (POM) samplers using parallel in situ and ex situ bioassays with homogenized untreated or activated carbon (AC) amended sediment. The AC amendment achieved a remedial efficiency in reducing bioaccumulation by 90% in the laboratory and by 44% in the field transplants. In situ measurements showed that PCB uptake by POM samplers was greater for POM placed in the surface sediment compared with the underlying AC amendment, suggesting that tidal exchange of surrounding material with similar PCB availability as untreated sediment was redeposited in the cages. Polychlorinated biphenyls bioaccumulation with caged polychaetes from untreated sediment was half as large under field conditions compared with laboratory conditions. A biodynamic model was used to confirm and quantify the different processes that could have influenced these results. Three factors appeared most influential in the bioassays: AC amendment significantly reduces bioavailability under laboratory and field conditions; sediment deposition within test cages in the field partially masks the remedial benefit of underlying AC-amended sediment; and deposit-feeders exhibit less PCB uptake from untreated sediment when feeding is reduced. Ex situ and in situ experiments inevitably show some differences that are associated with measurement methods and effects of the environment. Parallel ex situ and in situ bioassays, passive sampler measurements, and quantifying important processes with a model can tease apart these field influences. ?? 2010 SETAC.

  9. Innovative new method to test skill of bioassay in postgraduate students during doctor of medicine pharmacology examination using objective structured practical examination and animal experiment software


    Naresh D. Balani; Prabodh Anant Wankhade


    Background: Despite the guidelines for discontinuation of dissection and animal experimentation and introduction of use of alternatives to animal experimentation, Maharashtra University of Health Science, Nashik has not modified the exam pattern by shifting the emphasis on to use of animal experiment software. In one study on importance of bioassay in career, it has been reported that 96.6% did not use their knowledge of Bioassay during their 10 years of post MD career, whether in pharmaceuti...

  10. Comparison of methods of identifying Helicobacter hepaticus in B6C3F1 mice used in a carcinogenesis bioassay. (United States)

    Fox, J G; MacGregor, J A; Shen, Z; Li, X; Lewis, R; Dangler, C A


    In a long-term rodent bioassay evaluating the carcinogenicity of triethanolamine, there was equivocal evidence of carcinogenic activity in male B6C3F1 mice, based on a marginal increase in the number of hepatocellular adenomas and hepatoblastomas. Interpretation was complicated by the presence of Helicobacter hepaticus in selected silver-stained liver sections which also had histological evidence of karyomegaly and oval cell hyperplasia. An increase in numbers of liver tumors, as evidence of carcinogenic activity, was also noted in female mice. However, H. hepaticus was not considered a complicating factor, because the livers of the female mice did not have histological features compatible with H. hepaticus infection. A retrospective analysis of 51 liver tissue samples from the original carcinogenicity study was conducted to determine the incidence of H. hepaticus infection and to evaluate different diagnostic approaches for assessing the presence of H. hepaticus in livers lacking characteristic lesions. In an initial evaluation of seven mice with liver tumors, argyrophilic bacteria resembling H. hepaticus were observed in liver sections, associated with characteristic liver lesions of hepatocytic karyomegaly and oval cell hyperplasia. Frozen liver tissue was available from four of these mice; all were confirmed to be infected with H. hepaticus by culture and PCR. In a larger subsequent analysis using frozen liver tissues from 44 mice without characteristic hepatic lesions, H. hepaticus-specific DNA was amplified from the livers of 21 of 44 of the mice (47%), compared to 14 of 44 of the mice (32%) having H. hepaticus cultured from their frozen liver tumors. The results of H. hepaticus culture and H. hepaticus-specific PCR concurred (i.e., both positive and negative results) in 84% of the cases. Microscopic detection of immunofluorescence-labeled or silver-stained bacteria in liver sections was relatively insensitive compared to either culture or PCR detection. This

  11. Adulticidal and larvicidal activity of Beauveria bassiana and Metarhizium anisopliae against housefly, Musca domestica (Diptera: Muscidae), in laboratory and simulated field bioassays. (United States)

    Mishra, Sapna; Kumar, Peeyush; Malik, Anushree; Satya, Santosh


    The susceptibility of the adult and larval stage of housefly, Musca domestica L. (Diptera: Muscidae), to two entomopathogenic fungi, Metarhizium anisopliae (Metsch.) Sor. and Beauveria bassiana (Bals.) Vuill., was evaluated under laboratory and simulated field bioassays. Bioassays on adult houseflies were carried out at different conidial concentrations ranging from 10(3) to 10(9) conidia/ml in petri plate and minichamber assays. Absolute mortality was observed within 4-5 days at all the concentrations tested. M. anisopliae was found to be more effective with LC(50) of 6.75 × 10(7) conidia/ml compared with 1.21 × 10(8) conidia/ml of B. bassiana in petri plate bioassay. Similar trend was observed in minichamber bioassay. Larvicidal activity evaluated through petri plate bioassay also indicated that M. anisopliae was more effective larvicide with LC(50) of 4.1 × 10(8) conidia/ml as against 3.31 × 10(9) conidia/ml of B. bassiana. Larvicidal activity was further evaluated in simulated field condition of decaying waste matrix using dry conidial formulations (10(8) conidia/g) of both the fungi. Larval mortality obtained in this assay was 43% (B. bassiana) and 63% (M. anisopliae). Remarkably better performance of M. anisopliae as an adulticidal and larvicidal agent over B. bassiana in laboratory bioassays as well as simulated field conditions suggests that it may have good potential to become part of an integrated housefly control program.

  12. Use of a chemically induced-colon carcinogenesis-prone Apc-mutant rat in a chemotherapeutic bioassay

    Directory of Open Access Journals (Sweden)

    Yoshimi Kazuto


    Full Text Available Abstract Background Chemotherapeutic bioassay for colorectal cancer (CRC with a rat model bearing chemically-induced CRCs plays an important role in the development of new anti-tumor drugs and regimens. Although several protocols to induce CRCs have been developed, the incidence and number of CRCs are not much enough for the efficient bioassay. Recently, we established the very efficient system to induce CRCs with a chemically induced-colon carcinogenesis-prone Apc-mutant rat, Kyoto Apc Delta (KAD rat. Here, we applied the KAD rat to the chemotherapeutic bioassay for CRC and showed the utility of the KAD rat. Methods The KAD rat has been developed by the ENU mutagenesis and carries a homozygous nonsense mutation in the Apc gene (S2523X. Male KAD rats were given a single subcutaneous injection of AOM (20 mg/kg body weight at 5 weeks of age. Starting at 1 week after the AOM injection, they were given 2% DSS in drinking water for 7 days. Tumor-bearing KAD rats were divided into experimental and control groups on the basis of the number of tumors observed by endoscopy at week 8. The 5-fluorouracil (5-FU was administrated intravenously a dose of 50 or 75 mg/kg weekly at week 9, 10, and 11. After one-week interval, the 5-FU was given again at week 13, 14, and 15. At week 16, animals were sacrificed and tumor number and volume were measured macroscopically and microscopically. Results In total 48 tumors were observed in 27 KAD rats with a 100% incidence at week 8. The maximum tolerated dose for the KAD rat was 50 mg/kg of 5-FU. Macroscopically, the number or volume of tumors in the 5-FU treated rats was not significantly different from the control. Microscopically, the number of adenocarcinoma in the 5-FU treated rats was not significantly different (p Conclusion The use of the AOM/DSS-treated tumor-bearing KAD rats could shorten the experimental period and reduce the number of animals examined in the chemotherapeutic bioassay. The

  13. Rolling-based direct-transfer printing: A process for large-area transfer of micro- and nanostructures onto flexible substrates (United States)

    Grierson, D. S.; Flack, F. S.; Lagally, M. G.; Turner, K. T.


    A rolling-based printing approach for transferring arrays of patterned micro- and nano-structures directly from rigid fabrication substrates onto flexible substrates is presented. Transfer-printing experiments show that the new process can achieve high-yield and high-fidelity transfer of silicon nanomembrane components with diverse architectures to polyethylene terephthalate substrates over chip-scale areas (>1 × 1 cm2) in process are investigated through finite element simulations of the contact and transfer process. These mechanics models provide guidance for controlling the contact area and strain in the flexible substrate during transfer, both of which are key for achieving reproducible and controlled component transfer over large areas.

  14. The sensitivity of an hydroponic lettuce root elongation bioassay to metals, phenol and wastewaters. (United States)

    Park, Jihae; Yoon, Jeong-hyun; Depuydt, Stephen; Oh, Jung-Woo; Jo, Youn-min; Kim, Kyungtae; Brown, Murray T; Han, Taejun


    The root elongation bioassay is one of the most straightforward test methods used for environmental monitoring in terms of simplicity, rapidity and economy since it merely requires filter paper, distilled water and Petri dishes. However, filter paper as a support material is known to be problematic as it can reduce the sensitivity of the test. The newly developed hydroponic method reported here differs from the conventional root elongation method (US EPA filter paper method) in that no support material is used and the exposure time is shorter (48 h in this test versus 120 h in the US EPA test). For metals, the hydroponic test method was 3.3 (for Hg) to 57 (for Cu) times more sensitive than the US EPA method with the rank orders of sensitivity, estimated from EC50 values, being Cu≥Cd>Ni≥Zn≥Hg for the former and Hg≥Cu≥Ni≥Cd≥Zn for the latter methods. For phenol, the results did not differ significantly; EC50 values were 124 mg L(-1) and 108-180 mg L(-1) for the hydroponic and filter paper methods, respectively. Lettuce was less sensitive than daphnids to wastewaters, but the root elongation response appears to be wastewater-specific and is especially sensitive for detecting the presence of fluorine. The new hydroponic test thus provides many practical advantages, especially in terms of cost and time-effectiveness requiring only a well plate, a small volume of distilled water and short exposure period; furthermore, no specialist expertise is required. The method is simpler than the conventional EPA technique in not using filter paper which can influence the sensitivity of the test. Additionally, plant seeds have a long shelf-life and require little or no maintenance.

  15. Identification of estrogenic compounds in fish bile using bioassay-directed fractionation. (United States)

    Houtman, Corine J; Van Oostveen, Annemiek M; Brouwer, Abraham; Lamoree, Marja H; Legler, Juliette


    Conjugates of estrogenic chemicals, endogenous as well as xenobiotic, are mainly excreted via bile into the intestine. Therefore, measurement of estrogenic activity in bile yields useful information about an organism's internal exposure to (xeno-)estrogens. Although previous studies in The Netherlands have reported estrogenic activity in male fish bile, the contribution of natural hormones and xenobiotic substances to this activity is unknown. To identify compounds responsible for estrogenic activity in fish bile, we developed a bioassay-directed fractionation method for estrogenic chemicals. In this approach, the in vitro reporter gene assay ER-CALUX (Estrogen Responsive Chemical Activated Luciferase Gene Expression) was used to assess estrogenic activity in deconjugated bile samples and to direct RP-HPLC fractionation and chemical analysis (by GC-MS) of estrogenic compounds. The method was applied to bile from male breams (Abramis brama) collected at three locations in The Netherlands. At one of these locations, the River Dommel, extremely high levels of plasma vitellogenin and a high incidence of intersex gonads in these male breams have previously been observed, indicating the exposure to estrogens. In this study, the natural hormones 17beta-estradiol, estrone, and estriol accounted for the majority of estrogenic activity in male bream bile. At the River Dommel, the synthetic contraceptive pill component ethynylestradiol was found in effective concentrations as well. The detected natural and synthetic hormones may be responsible forthe estrogenic effects observed in wild bream from this location. Furthermore, a large number of xenobiotic chemicals was detected at relatively high levels in bile, including triclosan, chloroxylenol, and clorophene. Although chloroxylenol was shown for the first time to be weakly estrogenic, these compounds did not contribute significantly to the estrogenic activity observed.

  16. Bioassay-Guided Isolation of Sesquiterpene Coumarins from Ferula narthex Bioss: A New Anticancer Agent (United States)

    Alam, Mahboob; Khan, Ajmal; Wadood, Abdul; Khan, Ayesha; Bashir, Shumaila; Aman, Akhtar; Jan, Abdul Khaliq; Rauf, Abdur; Ahmad, Bashir; Khan, Abdur Rahman; Farooq, Umar


    The main objective of cancer management with chemotherapy (anticancer drugs) is to kill the neoplastic (cancerous) cell instead of a normal healthy cell. The bioassay-guided isolation of two new sesquiterpene coumarins (compounds 1 and 2) have been carried out from Ferula narthex collected from Chitral, locally known as “Raw.” Anticancer activity of crude and all fractions have been carried out to prevent carcinogenesis by using MTT assay. The n-hexane fraction showed good activity with an IC50 value of 5.434 ± 0.249 μg/mL, followed by crude MeFn extract 7.317 ± 0.535 μg/mL, and CHCl3 fraction 9.613 ± 0.548 μg/mL. Compounds 1 and 2 were isolated from chloroform fraction. Among tested pure compounds, compound 1 showed good anticancer activity with IC50 value of 14.074 ± 0.414 μg/mL. PASS (Prediction of Activity Spectra) analysis of the compound 1 was carried out, in order to predicts their binding probability with anti-cancer target. As a results the compound 1 showed binding probability with human histone acetyltransferase with Pa (probability to be active) value of 0.303. The compound 1 was docked against human histone acetyltransferase (anti-cancer drug target) by using molecular docking simulations. Molecular docking results showed that compound 1 accommodate well in the anti-cancer drug target. Moreover the activity support cancer chemo preventive activity of different compounds isolated from the genus Ferula, in accordance with the previously reported anticancer activities of the genus. PMID:26909039

  17. A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors. (United States)

    Stacker, Steven A; Halford, Michael M; Roufail, Sally; Caesar, Carol; Achen, Marc G


    The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

  18. Sharp-bounded zones link to the effect in planar chromatography-bioassay-mass spectrometry. (United States)

    Klingelhöfer, Ines; Morlock, Gertrud E


    The traditional direct bioautography workflow was substantially altered to yield narrow, sharp-bounded effective zones. For the first time, microorganisms quantitatively detected the single effective compounds in complex samples, separated in parallel on a planar chromatogram. This novel effect-directed workflow was demonstrated and optimized for the discovery of endocrine disrupting compounds (EDCs) reacting with the human estrogen receptor down to the femtogram-per-zone range, like 250fg/zone for 17β-estradiol (E2). For application volumes of up to 0.5mL, estrogen-effective compounds could directly be detected in complex samples at the ultratrace level (ng/kg-range). Sharp-bounded, estrogen-effective zones discovered were further characterized by direct elution into the mass spectrometer. HPTLC-ESI-MS mass spectra of (xeno)estrogens were shown for the first time. Owed to the substantially improved zone resolution, compound assignment was reliable and a comparison of the receptor affinities was conducted for six (xeno)estrogens. Also, long-term cell cultivation of the genetically modified yeast was demonstrated on the HPTLC plate. The optimized HPTLC-pYES workflow was proven for real food samples, exemplarily shown for beer. The general applicability of generating sharp-bounded zones was successfully proven by transfer of the fundamentally improved workflow to the Bacillus subtilis bioassay used for discovery of antibiotics in plant extracts. This new era of quantitative direct bioautography in combination with mass spectrometry will accelerate the scientific understanding in a wide application field via the streamlined access to fast and reliable information on effective components in complex samples.

  19. Application of a canine {sup 238}Pu dosimetry model to human bioassay data

    Energy Technology Data Exchange (ETDEWEB)

    Hickman, A.W. Jr. [Florida Univ., Gainesville, FL (United States)


    Associated with the use of 2{sup 238}Pu in thermoelectric power sources for space probes and power supplies for cardiac devices is the potential for human exposure to {sup 238}Pu, primarily by inhalation. In the event of human internal exposure, a means is needed for assessing the level of intake and calculating radiation doses. Several bioassay/dosimetry models have been developed for {sup 239}Pu. However, results from studies with laboratory animals have indicated that the biokinetics, and therefore the descriptive models, of {sup 238}Pu are significantly different from those for {sup 239}Pu. A canine model accounting for these differences has been applied in this work to urinary excretion data from seven humans occupationally exposed to low levels of an insoluble {sup 238}Pu compound. The modified model provides a good description of the urinary excretion kinetics observed in the exposed humans. The modified model was also used to provide estimates of the initial intakes of {sup 238}Pu for the seven individuals; these estimates ranged from 4.5 nCi (170 Bq) to 87 nCi (3200 Bq). Autopsy data on the amount and distribution of {sup 238}Pu retained in the organs may be used in the future to validate or refute both these estimates and the assumptions used to formulate the human model. Modification of the human model to simulate an injection exposure to {sup 239}Pu gave patterns of retention in the organs and urinary excretion comparable to those seen previously in humans; further modification of the model using fecal data (unavailable for the subjects of this study) is indicated.

  20. Bioassay studies support the potential for iatrogenic transmission of variant Creutzfeldt Jakob Disease through dental procedures.

    Directory of Open Access Journals (Sweden)

    Elizabeth Kirby

    Full Text Available BACKGROUND: Evidence is required to quantify the potential risks of transmission of variant Creutzfeldt Jakob (vCJD through dental procedures. Studies, using animal models relevant to vCJD, were performed to address two questions. Firstly, whether oral tissues could become infectious following dietary exposure to BSE? Secondly, would a vCJD-contaminated dental instrument be able to transmit disease to another patient? METHODS: BSE-301V was used as a clinically relevant model for vCJD. VM-mice were challenged by injection of infected brain homogenate into the small intestine (Q1 or by five minute contact between a deliberately-contaminated dental file and the gingival margin (Q2. Ten tissues were collected from groups of challenged mice at three or four weekly intervals, respectively. Each tissue was pooled, homogenised and bioassayed in indicator mice. FINDINGS: Challenge via the small intestine gave a transmission rate of 100% (mean incubation 157±17 days. Infectivity was found in both dental pulp and the gingival margin within 3 weeks of challenge and was observed in all tissues tested within the oral cavity before the appearance of clinical symptoms. Following exposure to deliberately contaminated dental files, 97% of mice developed clinical disease (mean incubation 234±33 days. INTERPRETATION: Infectivity was higher than expected, in a wider range of oral tissues, than was allowed for in previous risk assessments. Disease was transmitted following transient exposure of the gingiva to a contaminated dental file. These observations provide evidence that dental procedures could be a route of cross-infection for vCJD and support the enforcement of single-use for certain dental instruments.

  1. Trypanocidal Activity of Smallanthus sonchifolius: Identification of Active Sesquiterpene Lactones by Bioassay-Guided Fractionation

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    F. M. Frank


    Full Text Available In order to find novel plant-derived biologically active compounds against Trypanosoma cruzi, we isolated, from the organic extract of Smallanthus sonchifolius, the sesquiterpene lactones enhydrin (1, uvedalin (2, and polymatin B (3 by bioassay-guided fractionation technique. These compounds showed a significant trypanocidal activity against the epimastigote forms of the parasite with IC50 values of 0.84 μM (1, 1.09 μM (2, and 4.90 μM (3. After a 24 h treatment with 10 μg/mL of enhydrin or uvedalin, parasites were not able to recover their replication rate. Compounds 1 and 2 showed IC50 values of 33.4 μM and 25.0 μM against T. cruzi trypomastigotes, while polymatin B was not active. When the three compounds were tested against the intracellular forms of T. cruzi, they were able to inhibit the amastigote replication with IC50 of 5.17 μM, 3.34 μM, and 9.02 μM for 1, 2, and 3, respectively. The cytotoxicity of the compounds was evaluated in Vero cells obtaining CC50 values of 46.5 μM (1, 46.8 μM (2, and 147.3 μM (3 and the selectivity index calculated. According to these results, enhydrin and uvedalin might have potentials as agents against Chagas disease and could serve as lead molecules to develop new drugs.

  2. Allelopathy in a leguminous mangrove plant, Derris indica: protoplast co-culture bioassay and rotenone effect. (United States)

    Inoue, Aya; Mori, Daisuke; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako


    To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

  3. Bed rest suppresses bioassayable growth hormone release in response to muscle activity (United States)

    McCall, G. E.; Goulet, C.; Grindeland, R. E.; Hodgson, J. A.; Bigbee, A. J.; Edgerton, V. R.


    Hormonal responses to muscle activity were studied in eight men before (-13 or -12 and -8 or -7 days), during (2 or 3, 8 or 9, and 13 or 14 days) and after (+2 or +3 and +10 or +11 days) 17 days of bed rest. Muscle activity consisted of a series of unilateral isometric plantar flexions, including 4 maximal voluntary contractions (MVCs), 48 contractions at 30% MVC, and 12 contractions at 80% MVC, all performed at a 4:1-s work-to-rest ratio. Blood was collected before and immediately after muscle activity to measure plasma growth hormone by radioimmunoassay (IGH) and by bioassay (BGH) of tibia epiphyseal cartilage growth in hypophysectomized rats. Plasma IGH was unchanged by muscle activity before, during, or after bed rest. Before bed rest, muscle activity increased (P pattern that persisted through 8 or 9 days of bed rest. However, after 13 or 14 days of bed rest, plasma concentration of BGH was significantly lower after than before muscle activity (2,594 +/- 211 to 2,085 +/- 109 microg/l). After completion of bed rest, muscle activity increased BGH by 31% at 2 or 3 days (1,807 +/- 117 to 2,379 +/- 473 microg/l; P < 0.05), and by 10 or 11 days the BGH response was similar to that before bed rest (1,881 +/- 75 to 4,160 +/- 315 microg/l; P < 0.05). These data demonstrate that the ambulatory state of an individual can have a major impact on the release of BGH, but not IGH, in response to a single bout of muscle activity.

  4. Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-butyl ether in rats. (United States)

    Hagiwara, Akihiro; Doi, Yuko; Imai, Norio; Nakashima, Hironao; Ono, Takahiro; Kawabe, Mayumi; Furukawa, Fumio; Tamano, Seiko; Nagano, Kasuke; Fukushima, Shoji


    The modifying potential of ethyl tertiary-butyl ether (ETBE) on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay using male F344 rats. Animals were sequentially given 5 carcinogens with different target sites in the first 4 weeks for multi-organ initiation. After one week they received ETBE by gavage at dose levels of 0 (control), 300 or 1000mg/kg/day until experimental week 28. Further groups were also given ETBE at doses of 0 or 1000mg/kg/day without prior carcinogen application. Incidences and multiplicities of follicular cell hyperplasias and neoplasms in the thyroid were significantly increased at dose levels of more than 300mg/kg/day. Combined incidences of squamous cell hyperplasias and papillomas of the forestomach were also significantly increased at 300 and 1000mg/kg/day. Incidences and multiplicities of adenocarcinomas in the colon were increased at 1000mg/kg/day. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci per unit area of the liver sections, and the incidence of hepatocellular adenomas were also significantly increased at 1000mg/kg/day, along with multiplicities of atypical hyperplasias of renal tubules of the kidney and the incidence of papillomatosis of the urinary bladder. This latter lesion was also seen at low incidence at 1000mg/kg/day without initiation. Thus, the current results indicate that ETBE has tumor promoting potential for the thyroid and forestomach at dose levels of 300mg/kg/day and more, and for the colon, liver, kidney and urinary bladder at 1000mg/kg/day, under the present experimental conditions.

  5. Contact Bioassays with Phenoxybenzyl and Tetrafluorobenzyl Pyrethroids against Target-Site and Metabolic Resistant Mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sebastian Horstmann

    Full Text Available Mosquito strains that exhibit increased tolerance to the chemical class of compounds with a sodium channel modulator mode of action (pyrethroids and pyrethrins are typically described as "pyrethroid resistant". Resistance to pyrethroids is an increasingly important challenge in the control of mosquito-borne diseases, such as malaria or dengue, because one of the main interventions (the distribution of large numbers of long-lasting insecticide-treated bed nets currently relies entirely on long-lasting pyrethroids. Increasing tolerance of target insects against this class of insecticides lowers their impact in vector control. The current study suggests that the level of metabolic resistance depends on the structure of the molecule and that structurally different compounds may still be effective because detoxifying enzymes are unable to bind to these uncommon structures.Treated surface contact bioassays were performed on susceptible Aedes aegypti, East African knockdown resistance (kdr Anopheles gambiae (strain RSP-H and metabolically resistant Anopheles funestus (strain FUMOZ-R with different pyrethroids, such as cypermethrin, ß-cyfluthrin, deltamethrin, permethrin and transfluthrin (alone and in combination with the synergist piperonyl butoxide. The nonfluorinated form of transfluthrin was also assessed as a single agent and in combination with piperonyl butoxide.Although the dosages for pyrethroids containing a phenoxybenzyl moiety have exhibited differences in terms of effectiveness among the three tested mosquito species, the structurally different transfluthrin with a polyfluorobenzyl moiety remained active in mosquitoes with upregulated P450 levels. In trials with transfluthrin mixed with piperonyl butoxide, the added synergist exhibited no efficacy-enhancing effect.The results of this study suggest that transfluthrin has the potential to control P450-mediated metabolically resistant mosquitoes because the structural formula of

  6. Sample preparation method for the ER-CALUX bioassay screening of (xeno-)estrogenic activity in sediment extracts. (United States)

    Houtman, Corine J; Leonards, Pim E G; Kapiteijn, Wendy; Bakker, Joop F; Brouwer, Abraham; Lamoree, Marja H; Legler, Juliette; Klamer, Hans J C


    The application of bioassays to assess the occurrence of estrogenic compounds in the environment is increasing in both a scientific and statutory context. The availability of appropriate validated methods for sample pre-treatment and analysis is crucial for the successful implementation of bioassays. Here, we present a sample preparation method for the bioassay screening of estrogenic activity in sediment with the in vitro Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER-CALUX) assay. The method makes use of an Accelerated Solvent (ASE) or Soxhlet extraction with a mixture of dichloromethane and acetone (3:1, v/v), followed by clean up of the extract by Gel Permeation Chromatography (GPC). Recoveries of a panel of 17 pollutants differing largely in physical-chemical properties from spiked sediment were determined and appeared to be on average about 86%. Furthermore, the estrogenic potencies of all test compounds were individually assessed by determination of concentration-response relationships in the ER-CALUX assay. Concentration dependent estrogenic potency was found for 14 of the 17 compounds, with potencies of about 10(5) to 10(7) fold lower than the natural estrogenic hormone 17beta-estradiol. Anti-estrogenic potency was assessed by testing combinations of estradiol and individual test compounds, but was found for none of the compounds. The low estrogenic activity of the test compounds in the spiking mixture was well recovered during GPC treatment of the pure mixture, but did not contribute significantly to the background estrogenic activity present in the spiked sediment. Application of the method to field samples showed that estrogenic activity can be found at different types of locations, and demonstrated that levels between locations may vary considerably over relatively short distances.

  7. Ectomycorrhizal inoculum potential of northeastern US forest soils for American chestnut restoration: results from field and laboratory bioassays. (United States)

    Dulmer, Kristopher M; Leduc, Stephen D; Horton, Thomas R


    American chestnut (Castanea dentata) was once a dominant overstory tree in eastern USA but was decimated by chestnut blight (Cryphonectria parasitica). Blight-resistant chestnut is being developed as part of a concerted restoration effort to bring this heritage tree back. Here, we evaluate the potential of field soils in the northern portion of the chestnut's former range to provide ectomycorrhizal (EM) fungus inoculum for American chestnut. In our first study, chestnut seedlings were grown in a growth chamber using soil collected from three sites dominated by red oak (Quercus rubra) as inoculum and harvested after 5 months. Of the 14 EM fungi recovered on these seedlings, four species dominated in soils from all three sites: Laccaria laccata, a Tuber sp., Cenococcum geophilum, and a thelephoroid type. Seedlings grown in the nonsterilized soils were smaller than those growing in sterilized soils. In the second study, chestnut seedlings were grown from seed planted directly into soils at the same three sites. Seedlings with intermingling roots of established trees of various species were harvested after 5 months. Seventy-one EM fungi were found on the root tips of the hosts, with 38 occurring on chestnut seedlings. Multiple versus single host EM fungi were significantly more abundant and frequently encountered. The fungi observed dominating on seedlings in the laboratory bioassay were not frequently encountered in the field bioassay, suggesting that they may not have been active in mycelial networks in the field setting but were in the soils as resistant propagules that became active in the bioassay. These results show that soil from red oak stands can be used to inoculate American chestnut with locally adapted ectomycorrhizal fungi prior to outplanting, a relatively cost effective approach for restoration efforts.

  8. Rapid bioassay-guided screening of toxic substances in vegetable oils that shorten the life of SHRSP rats

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    Lewandowski Paul


    Full Text Available Abstract It has been consistently reported that vegetable oils including canola oil have a life shortening effect in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP and this toxic effect is not due to the fatty acid composition of the oil. Although it is possible that the phytosterol content or type of phytosterol present in vegetable oils may play some role in the life shortening effect observed in SHRSP rats this is still not completely resolved. Furthermore supercritical CO2 fractionation of canola oil with subsequent testing in SHRSP rats identified safe and toxic fractions however, the compounds responsible for life shortening effect were not characterised. The conventional approach to screen toxic substances in oils using rats takes more than six months and involves large number of animals. In this article we describe how rapid bioassay-guided screening could be used to identify toxic substances derived from vegetable oils and/or processed foods fortified with vegetable oils. The technique incorporates sequential fractionation of oils/processed foods and subsequent treatment of human cell lines that can be used in place of animal studies to determine cytotoxicity of the fractions with structural elucidation of compounds of interest determined via HPLC-MS and GC-MS. The rapid bioassay-guided screening proposed would require two weeks to test multiple fractions from oils, compared with six months if animal experiments were used to screen toxic effects. Fractionation of oil before bio-assay enhances the effectiveness of the detection of active compounds as fractionation increases the relative concentration of minor components.

  9. Hypoglycaemic activity of Gentiana olivieri and isolation of the active constituent through bioassay-directed fractionation techniques. (United States)

    Sezik, Ekrem; Aslan, Mustafa; Yesilada, Erdem; Ito, Shigeru


    Hypoglycemic effect of Gentiana olivieri Griseb. (Gentianaceae) flowering herbs on oral administration were studied using in vivo models in normal, glucose-hyperglycemic and streptozotocin-induced diabetic rats. Through in vivo bioassay-guided fractionation processes isoorientin, a known C-glycosylflavone, was isolated from the ethylacetate fraction by silica gel column chromatography as the main active ingredient from the plant. Isoorientin exhibited significant hypoglycemic and antihyperlipidemic effects at 15 mg/kg b.w.dose. Isoorientin concentration of the extracts and fractions were determined by HPLC in order to establish a correlation between the hypoglycaemic activity.

  10. Uranium in vitro bioassay action level used to screen workers for chronic inhalation intakes of uranium mill tailings. (United States)

    Reif, R H; Turner, J B; Carlson, D S


    A uranium in vitro bioassay (urinalysis) action level was derived for use at the Department of Energy's Uranium Mill Tailings Remedial Action Project sites to identify chronic inhalation intakes of uranium mill tailings causing 0.5 mSv (50 mrem) annual effective dose equivalent. All radionuclides in the 238U decay chain that contribute 1% or more to the annual effective dose equivalent from an inhalation intake of uranium mill tailings were included in the derivation of the urinalysis action level. Using a chronic inhalation intake model, the uranium urinalysis action level for a 24-h urine sample, collected on a quarterly schedule, was calculated to be 1.5 micrograms.

  11. Headspace-free setup of in vitro bioassays for the evaluation of volatile disinfection by-products. (United States)

    Stalter, Daniel; Dutt, Mriga; Escher, Beate I


    The conventional setup of in vitro bioassays in microplates does not prevent the loss of volatile compounds, which hampers the toxicological characterization of waterborne volatile disinfection by-products (DBPs). To minimize the loss of volatile test chemicals, we adapted four in vitro bioassays to a headspace-free setup using eight volatile organic compounds (four trihalomethanes, 1,1-dichloroethene, bromoethane, and two haloacetonitriles) that cover a wide range of air-water partition coefficients. The nominal effect concentrations of the test chemicals decreased by up to three orders of magnitude when the conventional setup was changed to a headspace-free setup for the bacterial cytotoxicity assay using bioluminescence inhibition of Vibrio fischeri. The increase of apparent sensitivity correlated significantly with the air-water partition coefficient. Purge and trap GC/MS analysis revealed a reduced loss of dosed volatile compounds in the headspace free setup (78-130% of nominal concentration) compared to a substantial loss in the conventional set up (2-13% of the nominal concentration). The experimental effect concentrations converged with the headspace-free setup to the effect concentrations predicted by a QSAR model, confirming the suitability of the headspace-free approach to minimize the loss of volatile test chemicals. The analogue headspace-free design of the bacterial bioassays for genotoxicity (umuC assay) and mutagenicity (Ames fluctuation assay) increased the number of compounds detected as genotoxic or mutagenic from one to four and zero to two, respectively. In a bioassay with a mammalian cell line applied for detecting the induction of the Nrf-2-mediated oxidative stress response (AREc32 assay), the headspace-free setup improved the apparent sensitivity by less than one order of magnitude, presumably due to the retaining effect of the serum components in the medium, which is also reflected in the reduced aqueous concentrations of compounds. This

  12. Human health risk assessment of soil dioxin/furans contamination and dioxin-like activity determined by ethoxyresorufin-O-deethylase bioassay. (United States)

    Man, Yu Bon; Chow, Ka Lai; Wang, Hong Sheng; Sun, Xiao Lin; Wu, Sheng Chun; Cai, Zong Wei; Kang, Yuan; Li, Hui; Zheng, Jin Shu; Giesy, John P; Wong, Ming Hung


    The major objective of this study was to evaluate the human health risks of agricultural land use conversion to other purposes in Hong Kong, based on the levels of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran (PCDD/Fs) and determined dioxin-like activity in soil using ethoxyresorufin-O-deethylase (EROD) bioassay. Hazard quotient showed soils of open burning site (OBS) and electronic waste open burning site (EW (OBS)) exert a relatively higher non-cancer risk on adults (50.9 and 8.00) and children (407 and 64.0) via the pathway of accidental ingestion of soil particles than other types of land use. In addition, the levels of 17 PCDD/Fs congeners in OBS and EW (OBS) soils indicated high and moderate (1654 and 260 in one million people) cancer risks through the above pathway. Furthermore, the biologically derived TCDD concentrations (TEQbio) were also significantly correlated to the chemically derived toxic equivalent concentrations of dioxin-like chemicals (TEQcal (sum of chemically derived 2,3,7,8-TeCDD toxic equivalent concentrations (TEQPCDD/F) and chemically derived dioxin-like PAHs toxic equivalent concentrations (TEQPAH)) (r = 0.770, p soil samples, indicating higher concentrations of PCDD/Fs derived from chemical analyses may reflect a higher potency of inducing EROD activity.

  13. Validation of a stability-indicating RP-LC method for the assessment of recombinant human interleukin-11 and its correlation with bioassay. (United States)

    Souto, Ricardo Bizogne; Stamm, Fernanda Pavani; Ribela, Maria Teresa de Carvalho Pinto; Bartolini, Paolo; Calegari, Guilherme Zanini; Dalmora, Sérgio Luiz


    A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 25°C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 µg/mL (r(2) = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.

  14. In vitro antiestrogenic effects of aryl methyl sulfone metabolites of polychlorinated biphenyls and 2,2-bis(4-chlorophenyl)-1,1-dichloroethene on 17beta-estradiol-induced gene expression in several bioassay systems. (United States)

    Letcher, Robert J; Lemmen, Josephine G; van der Burg, Bart; Brouwer, Abraham; Bergman, Ake; Giesy, John P; van den Berg, Martin


    Methylsulfonyl (MeSO(2)) metabolites of polychlorinated biphenyls (PCBs) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethene (4,4'-DDE), itself a metabolite of the insecticide 4,4'-DDT, are emerging as a major class of contaminants in the tissues of wildlife and humans. We investigated the antiestrogenic capacity and potencies of 3'- and 4'-MeSO(2)-2,2',4,5,5'-pentachlorobiphenyl (CB101) and -2,2',4,5'-tetrachlorobiphenyl (CB49), which are among the most environmentally persistent MeSO(2)-PCBs, and 3-MeSO(2)-4,4'-DDE on estrogen receptor (ER)-dependent gene expression in four cell-based bioassay systems. Congener- and concentration-dependent antagonism of 17beta-estradiol (E2)-induced gene expression, rather than induction of ER-dependent gene expression, was observed for the MeSO(2)-PCBs on lucifierase activity in stably transfected human breast adenocarcinoma T47D cells (ER-CALUX) and vitellogenin (vtg) production in primary hepatocytes from male carp fish (Cyprinus carpio) (CARP-HEP/vtg). 4'-MeSO(2)-CB101 and -CB49 had the highest antagonistic potency (i.e., maximum inhibition of about 70%, LOECs of 1.0 microM and 2.5 microM), whereas 3'-MeSO(2)-CB101 and -CB49 were less antagonistic; the precursor CB101 and MeSO(2)-PCB analog MeSO(2)-2,5-dichlorobenzene had no effect. Relative to the 4-MeSO(2)-PCBs, tamoxifen (IC(50), 0.06 microM and 0.7 microM) was about 40 and 7 times more potent in the ER-CALUX and CARP-HEP/vtg assays, respectively. Congener- and concentration-dependent effects on aryl hydrocarbon receptor-mediated induction of EROD activity (carp hepatocytes), luciferase expression (H4IIE rat hepatoma [H4IIE.luc] cell line), or cell viability were not observed. 3-MeSO(2)-4,4'-DDE was neither estrogenic nor antiestrogenic in either of the bioassays. Inhibitory trends for the MeSO(2)-PCBs in a bioassay based on stably transfected human embryonic kidney cell (HEK293-ERalpha-ERE) were similar to the ER-CALUX and CARP-HEP/vtg bioassays, whereas the antagonism was

  15. A new reliable bioassay for determining the biological activity of human interleukin-12 by using human NK cell line NKG cells. (United States)

    Cheng, Min; Fei, Baozhen; Zheng, Xiaodong; Chen, Yongyan; Sun, Rui; Wei, Haiming; Tian, Zhigang


    A specific and accurate bioassay for determining the biological activity of human interleukin-12 (hIL-12), an important typical Th1 cytokine in both innate and adaptive immunity, is extensively required for biomedical and clinical study. In this paper, we used a new established NK cell line NKG cells as the responder to hIL-12 stimulation by detecting their IFN-γ production. It was found that NKG cells produced high level of IFN-γ when simulated by hIL-12, and the dose-response curve became the best Sigmoid curve (R(2)=0.9977, pbioassay was precise and reproducible. Furthermore, no obvious cross-effects of other cytokines such as IL-2 and IL-18 was observed on the bioassay. Addition of the neutralizing anti-hIL-12 antibody to the bioassay significantly inhibited the IFN-γ production in a dose dependent manner, indicating that the bioactivity was actually mediated by hIL-12. The bioassay by using NKG cells was also suitable for determining the biological activity of recombinant hIL-12 in the form of purified product or culture supernatant by CHO-hIL-12 cell line. In conclusion, a reliable hIL-12 bioassay for determining its biological activity was established by using NKG cells as a responder and measuring their production of IFN-γ.

  16. A bioassay to evaluate the activity of chemical stimuli from grape berries on the oviposition of Lobesia botrana (Lepidoptera: Tortricidae). (United States)

    Maher, N; Thiéry, D


    A two-choice bioassay was developed to evaluate the role of host-plant berry compounds on the oviposition site acceptance of the generalist moth Lobesia botrana (Denis & Shiffermüller). A key feature was the lining of the bioassay arena with felt which focused oviposition on the test substrates. Initial experiments comparing substrates with different physical features indicated that smooth textures and spherical shapes with interstices favour oviposition. Artificial oviposition substrates were thus constructed with glass spheres in order to test the behavioural activity of grapevine berry extracts. Only polar extracts obtained by soaking berries in methanol or water stimulated oviposition (more eggs were laid on the extract-treated substrate than on the control substrate), whereas more apolar ones obtained with chloroform or hexane had no significant effect. The prior removal of epicuticular waxes from grape berries before extraction did not enhance the stimulatory activity of the methanol extract. The oviposition response to this extract was dose-dependent. It is concluded that polar compounds present on grape berries act as oviposition stimulants for L. botrana.

  17. Confirmatory chemical analyses and solid phase bioassays on sediment from the Columbia River Estuary at Tongue Point, Oregon

    Energy Technology Data Exchange (ETDEWEB)

    Young, J.S.; Word, J.Q.; Apts, C.W.; Barrows, M.E.; Cullinan, V.I.; Kohn, N.P.


    The Department of Economic Development, Ports Division, of the state of Oregon plans to develop a former ship supply and storage site near Tongue Point, Oregon, for commercial shipping. The development would require dredging the adjacent waterway to the Columbia River 40-foot channel to admit commercials vessels. The Portland District of the US Army Corps of Engineers requested the Battelle/Marine Sciences Laboratory (MSL) to conduct confirmatory solid-phase bioassays that would provide technical data for an evaluation of the potential environmental impact of ocean disposal of the dredged material. These confirmatory studies provided chemical and biological information required by ocean dumping regulations to determine suitability of Tongue Point sediments for ocean disposal. Sediment core samples were collected from Cathlamet Bay at Tongue Point in the upper Columbia River estuary. Sediment surface grab samples were collected at reference/control sites offshore from the mouth of the Columbia River (Disposal Site F) and at West Beach, Whidbey Island, Washington. The Tongue Point sediments were mixed into two composited batches. The MSL conducted solid-phase bioassays with these composites and reference sediments on four species of organisms.

  18. Bioassay-Guided Fractionation of a Leaf Extract from Combretum mucronatum with Anthelmintic Activity: Oligomeric Procyanidins as the Active Principle. (United States)

    Spiegler, Verena; Sendker, Jandirk; Petereit, Frank; Liebau, Eva; Hensel, Andreas


    Combretum mucronatum Schumach. & Thonn. is a medicinal plant widely used in West African traditional medicine for wound healing and the treatment of helminth infections. The present study aimed at a phytochemical characterization of a hydroalcoholic leaf extract of this plant and the identification of the anthelmintic compounds by bioassay-guided fractionation. An EtOH-H2O (1:1) extract from defatted leaves was partitioned between EtOAc and H2O. Further fractionation was performed by fast centrifugal partition chromatography, RP18-MPLC and HPLC. Epicatechin (1), oligomeric proanthocyanidins (OPC) 2 to 10 (mainly procyanidins) and flavonoids 11 to 13 were identified as main components of the extract. The hydroalcoholic extract, fractions and purified compounds were tested in vitro for their anthelmintic activity using the model nematode Caenorhabditis elegans. The bioassay-guided fractionation led to the identification of OPCs as the active compounds with a dose-dependent anthelmintic activity ranging from 1 to 1000 μM. Using OPC-clusters with a defined degree of polymerization (DP) revealed that a DP ≥ 3 is necessary for an anthelmintic activity, whereas a DP > 4 does not lead to a further increased inhibitory effect against the helminths. In summary, the findings rationalize the traditional use of C. mucronatum and provide further insight into the anthelmintic activity of condensed tannins.

  19. Bioassay-Guided Fractionation of a Leaf Extract from Combretum mucronatum with Anthelmintic Activity: Oligomeric Procyanidins as the Active Principle

    Directory of Open Access Journals (Sweden)

    Verena Spiegler


    Full Text Available Combretum mucronatum Schumach. & Thonn. is a medicinal plant widely used in West African traditional medicine for wound healing and the treatment of helminth infections. The present study aimed at a phytochemical characterization of a hydroalcoholic leaf extract of this plant and the identification of the anthelmintic compounds by bioassay-guided fractionation. An EtOH-H2O (1:1 extract from defatted leaves was partitioned between EtOAc and H2O. Further fractionation was performed by fast centrifugal partition chromatography, RP18-MPLC and HPLC. Epicatechin (1, oligomeric proanthocyanidins (OPC 2 to 10 (mainly procyanidins and flavonoids 11 to 13 were identified as main components of the extract. The hydroalcoholic extract, fractions and purified compounds were tested in vitro for their anthelmintic activity using the model nematode Caenorhabditis elegans. The bioassay-guided fractionation led to the identification of OPCs as the active compounds with a dose-dependent anthelmintic activity ranging from 1 to 1000 μM. Using OPC-clusters with a defined degree of polymerization (DP revealed that a DP ≥ 3 is necessary for an anthelmintic activity, whereas a DP > 4 does not lead to a further increased inhibitory effect against the helminths. In summary, the findings rationalize the traditional use of C. mucronatum and provide further insight into the anthelmintic activity of condensed tannins.

  20. Using a spiked sediment bioassay to establish a no-effect concentration for dioxin exposure to the amphipod, Ampelisca abdita

    Energy Technology Data Exchange (ETDEWEB)

    Barber, T.R.; Chappie, D.J.; Duda, D.J.; Fuchsman, P.C.; Finley, B.L. [ChemRisk--McLaren/Hart, Cleveland, OH (United States)


    A recent study conducted by the National Oceanic and Atmospheric Administration (NOAA) reported a highly significant correlation between 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) concentrations and amphipod (Ampelisca abdita) mortality in sediment samples collected from the lower Passaic River and Newark Bay. However, there is a paucity of data regarding the potential effects of 2,3,7,8-TCDD on benthic invertebrates under controlled laboratory exposures. In this study, 10-d whole-sediment bioassays using the marine amphipod A. abdita were conducted on spiked sediment samples representing a range of 2,3,7,8-TCDD concentrations. No effects on survival or growth relative to controls were observed at any test concentration. The highest 2,3,7,8-TCDD concentration reported from the NOAA study was 0.62 {micro}g/kg. Therefore, the lack of 2,3,7,8-TCDD toxicity in this study indicates that the mortality observed in the NOAA study was probably due to factors or chemicals other than 2,3,7,8-TCDD. This study demonstrates the utility of spiked sediment bioassays in evaluating cause and effect relationships between sediment contamination and benthic invertebrate mortality.

  1. Genotoxic and mutagenic effects of polluted surface water in the midwestern region of Brazil using animal and plant bioassays

    Directory of Open Access Journals (Sweden)

    Priscila Leocádia Rosa Dourado

    Full Text Available Abstract This study aimed to evaluate DNA damage in animal and plant cells exposed to water from the Água Boa stream (Dourados, Mato Grosso do Sul, Brazil by using bioassays, and to identify the chemical compounds in the water to determine the water quality in the area. Through the cytotoxicity bioassay with Allium cepa, using micronucleus test, and comet assay, using Astyanax altiparanae fish, the results indicated that biological samples were genetically altered. Micronuclei were observed in erythrocytes of A. altiparanae after exposure to water from locations close to industrial waste discharge. The highest DNA damage observed with the comet assay in fish occurred with the exposure to water from locations where the presence of metals (Cu, Pb, Cd, Ni was high, indicating the possibility of genotoxic effects of these compounds. Thus, these results reinforce the importance of conducting genotoxicity tests for developing management plans to improve water quality, and indicate the need for waste management before domestic and industrial effluents are released into the rivers and streams.

  2. Selection of a bioassay battery to assess toxicity in the affluents and effluents of three water-treatment plants

    Directory of Open Access Journals (Sweden)

    Paola Bohórquez-Echeverry


    Full Text Available The assessment of water quality includes the analysis of both physical-chemical and microbiological parameters. However,none of these evaluates the biological effect that can be generated in ecosystems or humans. In order to define the most suitable organismsto evaluate the toxicity in the affluent and effluent of three drinking-water treatment plants, five acute toxicity bioassays were used,incorporating three taxonomic groups of the food chain. Materials and methods. The bioassays used were Daphnia magna and Hydraattenuata as animal models, Lactuca sativa and Pseudokirchneriella subcapitata as plant models, and Photobacterium leioghnathi asbacterial model. To meet this objective, selection criteria of the organisms evaluated and cluster analysis were used to identify the mostsensitive in the affluent and effluent of each plant. Results. All organisms are potentially useful in the assessment of water quality bymeeting four essential requirements and 17 desirable requirements equivalent to 100% acceptability, except P. leioghnathi which doesnot meet two essential requirements that are the IC50 for the toxic reference and the confidence interval. The animal, plant and bacterialmodels showed different levels of sensitivity at the entrance and exit of the water treatment systems. Conclusions. H. attenuata, P.subcapitata and P. leioghnathi were the most effective organisms in detecting toxicity levels in the affluents and D. magna, P. subcapitataand P. leioghnathi in the effluents.

  3. Bioassay-guided extraction of crude fucose-containing sulphated polysaccharides from Sargassum fusiforme with response surface methodology (United States)

    Fu, Zhifei; Li, Haihua; Liu, Hongbing; Hu, Shuman; Li, Yueying; Wang, Mengxue; Guan, Huashi


    The response surface methodology (RSM) combined with bioassays was employed to optimize the extraction process of crude fucose-containing sulphated polysaccharides (cFCSP) from Sargassum fusiforme. The central composite design (CCD) was used with four variables, five levels, and four responses. The four variables were pH value of hydrochloric acid solution, extraction temperature (°C), ratio of liquid to raw material (mL g-1), and extraction time (h), respectively. Chemical and bioassay indices were used in combination as the response parameters, which included the yield of cFCSP, fucose content, proliferation rate of spleen cells, and lipopolysaccharide-induced proliferation of splenocytes. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis, and examined using the appropriate statistical methods. The best extraction conditions were as follows: the pH value of hydrochloric acid solution was 3.50; the extraction temperature was 100°C; the ratio of liquid to raw material was 15.00 mL g-1 and the extraction time was 2.50 h. The experimental yield was close to the predicted from the model. The extract could promote spleen lymphocyte proliferation, especially the lipopolysaccharide-induced lymphocyte proliferation in vitro, which suggested that its immunomodulatory effect on B lymphocytes. Therefore, cFCSP extracted from S. fusiforme could be utilized as an immunostimulant in functional foods and pharmaceutical industry in future.

  4. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. (United States)

    Caldwell, Gary S; Bentley, Matthew G; Olive, Peter J W


    Water soluble algal extracts, the aldehydes 2E,4E-decadienal, decanal, undecanal and the fatty acid eicosapentaenoic acid (EPA) were assayed for toxicity to hatching success and larval mortality of the brine shrimp Artemia salina. Both crude cellular extracts of the diatoms Skeletonema costatum and Nitzschia commutata and the diatom-derived short chain aldehyde decadienal were found to inhibit hatching success of A. salina cysts in a dose-dependent manner. Decadienal also significantly affected larval mortality rates in 24 and 72 h exposure incubations. The Artemia hatching success assay was the least sensitive of the three (EC50=3.94 microg ml(-1)). A greater sensitivity was observed for the 72 h compared with the 24 h exposure trials (EC50 for 24h=2.14, 72 h=0.023 microg ml(-1)). Decanal did not significantly affect survival or hatching success at the concentrations tested. Undecanal and EPA showed a limited toxic effect in naupliar mortality trials. We suggest that 72 h Artemia exposure trials represent an acceptable bioassay for diatom toxicity where alternative bioassays are unavailable.

  5. Bioanalytical screening methods for dioxins and dioxin-like compounds a review of bioassay/biomarker technology. (United States)

    Behnisch, P A; Hosoe, K; Sakai, S


    Determination of environmental pollutants utilizing biodetectors such as bioassays, biomarkers, enzyme immunoassays (EIAs), or other bioanalytical tools is a continuously growing area. The present literature review describes the principles and advantages/limitations of several bioanalytical detection methods (BDMs) for the screening and diagnosis of dioxin and dioxin-like compounds. This study characterizes briefly the family of dioxin and dioxin-like compounds, discusses potential Ah receptor (AhR) ligands and cytochrome P-450 (CYP) 1A1-enzyme-inducing compounds. 'Milestones' in the development of BDMs are summarized and explained in detail for a number of bioanalytical tools that can be used to detect these classes of dioxin-like persistent bioaccumulative toxicants (PBTs). The design of a screening profile with a battery of bioassays/biomarkers coupled with the chemical analysis is evaluated. The relative potencies (REPs) to 2,3,7,8-TCDD for dioxin-like compounds are reviewed for various BDMs and the differences are noted.

  6. Acute toxicity of drainage ditch water from a Washington State cranberry-growing region to Daphnia pulex in laboratory bioassays. (United States)

    Wood, Barbara; Stark, John D


    High concentrations of organophosphorous insecticides resulting from cranberry bog applications were detected in the Grayland Drainage Ditch (GDD) system in Grayland, Washington State, during the 1994-1996 Washington State Department of Ecology Pesticide Monitoring Program. This drainage ditch system drains cranberry bogs and enters the Pacific Ocean via the North Cove and Supon Inlet. Concerns about the impact of these pesticides on human and environmental health led to this investigation of the potential impact on an indicator species, Daphnia pulex. To determine the toxic effects of multiple pesticides entering the GDD, standardized laboratory toxicity tests with D. pulex were conducted concurrently with the Washington State Department of Ecology pesticide sampling. Concentrations of three insecticides, diazinon, chlorpyrifos, and azinphosmethyl, were the highest ever detected in state waters. The GDD water was found to cause acute toxicity in 33% of the laboratory bioassays conducted. Regression analysis, however, detected a poor correlation between total insecticide detected and percentage mortality of D. pulex at the two drainage ditch sites studied, Grays Harbor County site and the Pacific County site. However, the relationship between mortality of D. pulex and detected concentrations of diazinon and chlorpyrifos were significant. Sampling schedules for chemical analysis and bioassay testing appear to be the primary reason that statistical analysis failed to correlate mortality with detected OP pesticide concentrations. Grab samples used in toxicity testing may over- or underestimate actual concentrations of contaminants present in the system being studied.

  7. Study of the antimicrobial activity of metal complexes and their ligands through bioassays applied to plant extracts

    Directory of Open Access Journals (Sweden)

    Antonio F. Santos


    Full Text Available The appearance of resistant bacteria was found to reduce the efficiency of antimicrobial therapies with the current antibiotics, thereby increasing the need for more efficient drugs for the treatment of infections. Several studies have demonstrated an increase in antimicrobial activity following the interaction of several compounds with metal ions. The present study used a methodology adapted for antimicrobial bioassays using plant extracts, in compliance with the standards of the Clinical and Laboratory Standards Institute against Gram-positive and Gram-negative bacteria. The results obtained were considered appropriate for determining MIC, MBC as for performing antimicrobial sensitivity testing with good efficiency and reproducibility. The bacteriaPseudomonas fluorescens exhibited high sensitivity to the tested compounds, being efficient to evaluate the antibacterial activity. The bioassays with the metal complexes of flavonoid quercetin and Ga(III ions, and synthetic ligand H2bbppd and Cu(II ions showed a greater inhibitory effect than their individual ligands, thus, the addition indicated an increase in the antimicrobial activity after the coordination. Both metal complexes exhibit good antimicrobial performances, such as low minimum inhibitory concentration (MIC ≤ 250 µg/ml, bactericidal effect and a broad activity spectrum, which qualify these compounds as suitable candidates to the next step of drugs fabrication. Nevertheless, further studies on the mechanism of growth inhibition and toxicity are needed, in order to evaluate the potential of therapeutic application.

  8. Comparative surfactant reactivity of canine and human stratum corneum: a plea for the use of the corneosurfametry bioassay. (United States)

    Goffin, V; Fontaine, J; Piérard, G E


    Comparative dermatology has paid little attention to the physiopathology of the stratum corneum. In this study, we investigated the responses of human and canine horny layers to marketed animal wash products by using the corneosurfametry bioassay. Previous work has shown that, with increasing surfactant aggressiveness to the stratum corneum, the colorimetric index of mildness (CIM) decreases, while both the corneosurfametry index (CSMI) and the overall difference in corneosurfametry (ODC) increase. In the present study, stratum corneum reactivity to wash products and inter-individual variability were significantly higher in humans than in dogs. For the three corneosurfametry variables, linear correlations were found between data gathered in the two panel groups. In conclusion, this pilot study suggests that mean stratum corneum reactivity to surfactants is stronger in humans than in dogs. Inter-individual variation, indicative of sensitive skin, also appears to be broader in humans. As a consequence, data gathered from dogs by using the corneosurfametry bioassay cannot be extrapolated to humans. Such variation between species could be important in the assessment of product safety and in supporting claims for mildness.

  9. Evaluation of a combined macrophyte-epiphyte bioassay for assessing nutrient enrichment in the Portneuf River, Idaho, USA. (United States)

    Ray, Andrew M; Mebane, Christopher A; Raben, Flint; Irvine, Kathryn M; Marcarelli, Amy M


    We describe and evaluate a laboratory bioassay that uses Lemna minor L. and attached epiphytes to characterize the status of ambient and nutrient-enriched water from the Portneuf River, Idaho. Specifically, we measured morphological (number of fronds, longest surface axis, and root length) and population-level (number of plants and dry mass) responses of L. minor and community-level (ash-free dry mass [AFDM] and chlorophyll a [Chl a]) responses of epiphytes to nutrient enrichment. Overall, measures of macrophyte biomass and abundance increased with increasing concentrations of dissolved phosphorus (P) and responded more predictably to nutrient enrichment than morphological measures. Epiphyte AFDM and Chl a were also greatest in P-enriched water; enrichments of N alone produced no measurable epiphytic response. The epiphyte biomass response did not directly mirror macrophyte biomass responses, illustrating the value of a combined macrophyte-epiphyte assay to more fully evaluate nutrient management strategies. Finally, the most P-enriched waters not only supported greater standing stocks of macrophyte and epiphytes but also had significantly higher water column dissolved oxygen and dissolved organic carbon concentrations and a lower pH. Advantages of this macrophyte-epiphyte bioassay over more traditional single-species assays include the use of a more realistic level of biological organization, a relatively short assay schedule (~10 days), and the inclusion of multiple biological response and water-quality measures.

  10. Evaluation of a combined macrophyte–epiphyte bioassay for assessing nutrient enrichment in the Portneuf River, Idaho, USA (United States)

    Ray, Andrew M.; Mebane, Christopher A.; Raben, Flint; Irvine, Kathryn M.; Marcarelli, Amy M.


    We describe and evaluate a laboratory bioassay that uses Lemna minor L. and attached epiphytes to characterize the status of ambient and nutrient-enriched water from the Portneuf River, Idaho. Specifically, we measured morphological (number of fronds, longest surface axis, and root length) and population-level (number of plants and dry mass) responses of L. minor and community-level (ash-free dry mass [AFDM] and chlorophyll a [Chl a]) responses of epiphytes to nutrient enrichment. Overall, measures of macrophyte biomass and abundance increased with increasing concentrations of dissolved phosphorus (P) and responded more predictably to nutrient enrichment than morphological measures. Epiphyte AFDM and Chl a were also greatest in P-enriched water; enrichments of N alone produced no measurable epiphytic response. The epiphyte biomass response did not directly mirror macrophyte biomass responses, illustrating the value of a combined macrophyte–epiphyte assay to more fully evaluate nutrient management strategies. Finally, the most P-enriched waters not only supported greater standing stocks of macrophyte and epiphytes but also had significantly higher water column dissolved oxygen and dissolved organic carbon concentrations and a lower pH. Advantages of this macrophyte–epiphyte bioassay over more traditional single-species assays include the use of a more realistic level of biological organization, a relatively short assay schedule (~10 days), and the inclusion of multiple biological response and water-quality measures.

  11. Assessment of the potential toxicity of a linear alkylbenzene sulfonate (LAS) to freshwater animal life by means of cladoceran bioassays. (United States)

    Coelho, Katiuscia da Silva; Rocha, Odete


    The acute and chronic toxic effects of LAS on the cladocerans Daphnia similis, Ceriodaphnia dubia and Ceriodaphnia silvestrii were tested. Both types of toxicity bioassays and the methods of culture and stock maintenance of the test organisms conformed to the recommendations of ABNT (Brazilian Society of Technical Standards), which closely follow the standard methods of USEPA. The results obtained for EC(50) (48 h) were: 14.17 mg L(-1) for D. similis, 11.84 mg L(-1) for C. dubia and 13.52 mg L(-1) for C. silvestrii. In the chronic toxicity tests performed on C. dubia and C. silvestrii, there was a significant decrease in the fecundity of the exposed animals; the value of NOEC for C. dubia and C. silvestrii were 1.00 mg L(-1) and 2.50 mg L(-1), respectively. Cladoceran bioassays provided evidence that LAS concentration as low as 1.00 mg L(-1) can damage invertebrate animal life in freshwaters, concentrations that can be found in many eutrophic rivers and reservoirs.

  12. Bioassay technique for Plutella xylostella: Leaf-dip method%小菜蛾抗药性监测方法——叶片药膜法

    Institute of Scientific and Technical Information of China (English)

    郭磊; 边全乐; 张宏军; 高希武; 梁沛


    The diamondback moth, Plutella xylostella ( L. ) , is a notorious pest insect of cruciferous vegetables. Bioassays are an important technique for insecticide resistance monitoring and for screening the effectiveness of insecticides. This paper describes in detail the extensively used leaf dipping method in the DBM bioassay so as to provide a standard bioassay method for DBM and other lepidopterous pest insects.%小菜蛾Plutella xylostella(L.)是重要的十字花科蔬菜害虫.生物测定是进行害虫抗药性监测及筛选有效防治药剂的重要技术.本文对小菜蛾的生物测定技术进行了详细总结,以期为小菜蛾等鳞翅目害虫幼虫生物测定方法的规范提供依据.

  13. Investigations on sediment toxicity of German rivers applying a standardized bioassay battery. (United States)

    Hafner, Christoph; Gartiser, Stefan; Garcia-Käufer, Manuel; Schiwy, Sabrina; Hercher, Christoph; Meyer, Wiebke; Achten, Christine; Larsson, Maria; Engwall, Magnus; Keiter, Steffen; Hollert, Henner


    River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01% (standard) up to 0.25% (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l(-1). The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an ErC50 (72 h) of 0.00226 g l(-1) SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l(-1) SEQ. corresponding to 0.01% DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05% dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB

  14. Development of bioassay for pathogenecity testing of Ureaplasma urealyticum as part of host-pathogen communication

    Directory of Open Access Journals (Sweden)

    Purnomo Soeharso


    Full Text Available Bioassay of Ureaplasma urealyticum is necessary for detection as well as determination of pathogenic factors in order to understand the pathogenesis of diseases associate with ureaplasma infection. Cultivation and verification of ureaplasma is the first step of this study in the purpose of discovering sensitive method for ureaplasma detection. Cultivation of ureaplasma either in liquid or in solid media are able to detect the existence of ureaplasma in samples analyzed. However, application of PCR using specific primers to be compatible with urease gene (ure would confirm the presence of ureaplasma. The pathogenicity of ureaplasma is potentially monitored using reporter gene as a marker for gene expression. IceC was chosen as reporter gene for ureaplasma pathogenic determination as the gene has great sensitivity, easily detectable and quantitated in simple method of ice nucleation assay. Transposon 916 (Tn916 was selected as a vector for iceC gene to transform ureaplasma. The application of recombinant Tn916-iceC which is considered as pUI, allow detection of ureaplasma activities when transform ureaplasma is tested by ice nucleation assay. It was expected that ureaplasma transformation is the manifestation of mutagenesis which interfere genes responsible for bacterial pathogenicity, in order pathogenesis of bacterial infection to be analyzed accurately. IgA1 protease is considered to be an important factor for ureaplasma pathogenicity as the enzyme is required for successful colonization. Identification of iga gene and  determination of IgA1 protease activity are important for understanding the pathogenesis of ureaplasma infection. Putative iga gene of Mycoplasma genitalium was used as a reference to identify the presence of iga nucleotide sequence in U. urealyticum. Convincing evidence were obtained after PCR amplification of ureaplasma DNA using primers designed to be compatible with putative iga gene of M. genitalium followed by the

  15. Assessing HTS performance using BioAssay Ontology: screening and analysis of a bacterial phospho-N-acetylmuramoyl-pentapeptide translocase campaign. (United States)

    Moberg, Andreas; Zander Balderud, Linda; Hansson, Eva; Boyd, Helen


    With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.

  16. Integration of Microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products. (United States)

    Bohni, Nadine; Cordero-Maldonado, María Lorena; Maes, Jan; Siverio-Mota, Dany; Marcourt, Laurence; Munck, Sebastian; Kamuhabwa, Appolinary R; Moshi, Mainen J; Esguerra, Camila V; de Witte, Peter A M; Crawford, Alexander D; Wolfender, Jean-Luc


    Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation - generic for NP extracts of diverse origin - that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay

  17. Two Novel Bioassays to Assess the Effects of Pyrethroid-Treated Netting on Knockdown-Susceptible Versus Resistant Strains of Aedes aegypti. (United States)

    Denham, Steven; Eisen, Lars; Beaty, Meaghan; Beaty, Barry J; Black, William C; Saavedra-Rodriguez, Karla


    We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel.

  18. General principles and practice for insecticide bioassays%杀虫药剂生物测定原理与实践

    Institute of Scientific and Technical Information of China (English)

    辛娟娟; 高希武


    General principles, the selection of pest targets, standardization of assay procedures and biological statistics for the analyses of insecticide bioassays are reviewed. This review will provide a reference for developing standard insecticide bioassays.%本文从生物测定的基本原理、试验靶标的选择、杀虫药剂毒力测定标准化和统计分析等方面对杀虫药剂的毒力测定进行了规范性综述,为进行标准的生物测定提供依据和参考.


    Institute of Scientific and Technical Information of China (English)

    张金艳; 曲虹云; 杨青


    本文详细介绍了抑制光合作用除草剂的作用原理和除草剂的生物测定方法,主要介绍几种常用的抑制光合作用除草剂的生物测定方法并举例说明。%This paper introduces the action principles of herbicides with inhibited photosynthesis and the bioassay methods of herbicides, mainly introduces several the bioassay methods of the inhibited photosynthesis herbicides, and illustrates for instance。

  20. Seasonal changes in food quantity and quality of the common North Sea copepods Temora longicornis and Pseudocalanus elongatus: a bioassay approach

    DEFF Research Database (Denmark)

    Koski, Marja; Dutz, Jörg; Klein Breteler, W.;


    We evaluated the food quantity and quality over a seasonal cycle for the development and egg production of the common North Sea copepods Temora longicornis and Pseudocalanus elongatus, using a bioassay approach. Seston was sampled from December to October from a well-mixed water column of the Mar......We evaluated the food quantity and quality over a seasonal cycle for the development and egg production of the common North Sea copepods Temora longicornis and Pseudocalanus elongatus, using a bioassay approach. Seston was sampled from December to October from a well-mixed water column...

  1. 内分泌干扰物生物检测研究进展%Review of Bioassay Methods of Environmental Endocrine Disruptors Detection

    Institute of Scientific and Technical Information of China (English)



    The problem of environmental endocrine disruptors has become a global issue.Several bioassay methods of endocrine disruptors are overviewed.The trends of bioassay methods are prospected.%介绍了发光细菌法、重组基因酵母法、免疫分析法、细胞增殖法、受体结合活性法、卵黄蛋白法、酵母双杂交法等几种环境中内分泌干扰物的生物检测技术及研究进展,并提出了展望。

  2. Characterization of estrogen and androgen activity of food contact materials by different in vitro bioassays (YES, YAS, ERα and AR CALUX) and chromatographic analysis (GC-MS, HPLC-MS). (United States)

    Mertl, Johannes; Kirchnawy, Christian; Osorio, Veronica; Grininger, Angelika; Richter, Alexander; Bergmair, Johannes; Pyerin, Michael; Washüttl, Michael; Tacker, Manfred


    Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results.

  3. Confirmatory sediment analyses and solid and suspended particulate phase bioassays on sediment from Oakland Inner Harbor, San Francisco, California

    Energy Technology Data Exchange (ETDEWEB)

    Word, J.Q.; Ward, J.A.; Apts, C.W.; Woodruff, D.L.; Barrows, M.E.; Cullinan, V.I.; Hyland, J.L.; Campbell, J.F.


    The US Army Corps of Engineers (USACE), San Francisco District, was authorized by the US Congress to deepen the navigation channels of Inner and Outer Oakland Harbor, California. During review of the environmental impact statement required for this dredging and disposal project, a panel of national experts approved the open-water disposal of dredged sediment from selected areas within the Inner Harbor, subject to results of confirmatory solid phase bioassays. The San Francisco District of the Corps requested the Battle/Marine Sciences Laboratory (MSL) to conduct these confirmatory studies. The studies provided technical data for an evaluation of the potential environmental impact of this project. Within extremely narrow time constraints, these studies provided chemical and biological information required by ocean dumping regulations to determine suitability of the Oakland Inner Harbor and turning basin sediment for ocean disposal. 23 refs., 18 figs., 45 tabs.

  4. Phytotoxicity and Cytotoxicity of Essential Oil from Leaves of Plectranthus amboinicus, Carvacrol, and Thymol in Plant Bioassays. (United States)

    Pinheiro, Patrícia Fontes; Costa, Adilson Vidal; Alves, Thammyres de Assis; Galter, Iasmini Nicoli; Pinheiro, Carlos Alexandre; Pereira, Alexandre Fontes; Oliveira, Carlos Magno Ramos; Fontes, Milene Miranda Praça


    The essential oil of Plectranthus amboinicus and its chemotypes, carvacrol and thymol, were evaluated on the germination and root and aerial growth of Lactuca sativa and Sorghum bicolor and in acting on the cell cycle of meristematic root cells of L. sativa. The main component found in the oil by analysis in gas chromatography-mass spectrometry and gas chromatography flame ionization detection was carvacrol (88.61% in area). At a concentration of 0.120% (w v(-1)), the oil and its chemotypes retarded or inhibited the germination and decreased root and aerial growth in monocot and dicot species used in the bioassays. In addition, all substances caused changes in the cell cycle of the meristematic cells of L. sativa, with chromosomal alterations occurring from the 0.015% (w v(-1)) concentration. The essential oil of P. amboinicus, carvacrol, and thymol have potential for use as bioherbicides.

  5. A novel bioassay for high-throughput screening microorganisms with N-acyl homoserine lactone degrading activity. (United States)

    Liu, Pengfu; Gao, Yang; Huang, Wei; Shao, Zongze; Shi, Jiping; Liu, Ziduo


    A novel biosensor strain (Escherichia coli ALM403) that responded to N-acyl homoserine lactone (AHL) was constructed using a luxR-Plux cassette as a regulatory sequence and β-mannanase as a reporter gene. Dinitrosalicylic acid method was used to detect the response of the sensor strain to N-acyl homoserine lactone. By investigating the response to a range of concentrations of N-β-oxooctanoyl-L-homoserine lactone (OOHL), it was demonstrated that the expression of mannanase in E. coli ALM403 could be greatly enhanced by OOHL and resulted in an assayable phenotype. A high-throughput screening approach was developed to isolate AHL-degrading microorganisms, and a marine Halomonas sp. S66-4 showing a marked AHL-degrading ability was successfully isolated. In conclusion, the bioassay system provided a simple and efficient approach to isolate AHL-degrading bacteria.

  6. Assessment of Sediment Heavy Metals Pollution Using Screening Methods (XRF, TGA/MS, XRPD and Earthworms Bioassay) (United States)

    Findoráková, Lenka; Šestinová, Ol'ga; Hančul'ák, Jozef; Fedorová, Erika; Zorkovská, Anna


    The aim of this study is focused on the use of screening methods (TG/DTA coupled with MS, XRF, AAS, XRPD and earthworm bioassay) for sediments pollution assessing by heavy metals (Cu, Zn, Pb, Hg) coming from the former mining workloads in the central Spis, Eastern Slovakia. The screening methods (XRF, AAS) indicated pollution of studied sediments by Cu, Zn, Pb, Hg. The earthworms Dendrobaena veneta caused in some studied samples decrease of heavy metals concentration after their 7 days’ exposure in sediments. The other screening methods such as thermal analysis and XRPD analysis, does not confirm the specifically changes in physicochemical properties comparing the properties before and after 7 days’ earthworm's exposure.

  7. Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concen