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Applied and Environmental Microbiology Dec 2019Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and...
Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a mutant of defective in guanine biosynthesis. When supplemented with a plasmid expressing the genes for an -demethylation pathway from CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (, , and ). We show that strains of containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations. Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.
Topics: Biological Assay; Caffeine; Escherichia coli; Pseudomonas putida; Xanthines
PubMed: 31540989
DOI: 10.1128/AEM.01965-19 -
Marine Drugs Dec 2018Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place...
Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.
Topics: Animal Testing Alternatives; Animals; Biological Assay; Bivalvia; Cell Line, Tumor; Chromatography, High Pressure Liquid; False Positive Reactions; Marine Toxins; Mice; Mollusk Venoms; Oxocins; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry
PubMed: 30545061
DOI: 10.3390/md16120501 -
Cytometry. Part a : the Journal of the... Apr 2009
Topics: Animals; Biological Assay; Cell Communication; Cell Shape; Cell Size; Cytological Techniques; Humans; Image Cytometry; Microscopy; Software
PubMed: 19296510
DOI: 10.1002/cyto.a.20719 -
SLAS Discovery : Advancing Life... Dec 2021Well-behaved, in vitro bioassays generally produce normally distributed values in their primary (efficacy) data. Accordingly, the best practices for statistical analysis...
Well-behaved, in vitro bioassays generally produce normally distributed values in their primary (efficacy) data. Accordingly, the best practices for statistical analysis are well documented and understood. However, assays may occasionally display unusually high variability and fall outside the assumptions inherent in these standard analyses. These assays may still be in the optimization phase, in which the source of variation could be identified and addressed. They might also represent the best available option to address the biological process being examined. In these cases, the use of robust statistical methods may provide a more appropriate set of tools for both data analysis and assay optimization. This article provides guidance on best practices for the use of robust statistical methods for the analysis of bioassay data as an alternative to standard methods. Impacts on experimental design and interpretation will be discussed.
Topics: Biological Assay
PubMed: 34474612
DOI: 10.1177/24725552211038379 -
The Science of the Total Environment Nov 2019Publicly available chemical assessments of hydraulic fracturing related waters are generally based on shale gas practices in the U.S. There is a lack of information on...
Publicly available chemical assessments of hydraulic fracturing related waters are generally based on shale gas practices in the U.S. There is a lack of information on hydraulic fracturing related gas development from EU countries and more generally on other types of extractions. This research fills this knowledge gap by presenting chemical and bioassay assessments of hydraulic fracturing related waters from a tight gas development in the Netherlands. Fracturing fluid, flowback water and groundwater from surrounding aquifers before and after the actual fracturing were analysed by means of high resolution liquid chromatography tandem mass spectrometry, the Ames test and three chemical activated luciferase gene expression bioassays aimed at determining genotoxicity, oxidative stress response and polyaromatic hydrocarbon contamination. After sample enrichment a higher number of peaks can be found in both fracturing fluid and flowback samples. No clear differences in chemical composition were shown in the groundwater samples before and after hydraulic fracturing. Preliminary environmental fate data of the tentatively identified chemicals points towards persistence in water. Clear genotoxic and oxidative stress responses were found in the fracturing fluid and flowback samples. A preliminary suspect screening resulted in 25 and 36 matches in positive and negative ionisation respectively with the 338 possible suspect candidates on the list. Extensive measures relating to the handling, transport and treatment of hydraulic fracturing related waters are currently in place within the Dutch context. The results of the present study provide a scientific justification for such measures taken to avoid adverse environmental and human health impacts.
Topics: Biological Assay; Environmental Monitoring; Hydraulic Fracking; Netherlands; Oil and Gas Fields; Water Pollutants, Chemical
PubMed: 31301504
DOI: 10.1016/j.scitotenv.2019.06.354 -
Regenerative Medicine May 2020The field of cell therapy has blossomed, providing exciting new options for treating a variety of diseases. While few cell therapy products have US FDA approval, there... (Review)
Review
The field of cell therapy has blossomed, providing exciting new options for treating a variety of diseases. While few cell therapy products have US FDA approval, there are thousands of cell treatments at various stages of development, pointing to a potential revolutionary shift in patient care. The expanding number and nature of cellular therapies necessitate greater standardization. Several international organizations are collaborating to pursue some level of global standardization, especially concerning cell banking. However, less harmonization surrounds assays used for critical quality characterization including: identity, purity, safety and potency. Frequently, there is divergence regarding the terms describing the characterization assays across regulatory authorities and guidances. This review summarizes the critical quality assays currently used for different categories of cell therapies. Areas of harmonization and an absence of standardization are highlighted. We propose potential solutions to facilitate harmonization of critical quality characterization assays and the language used to describe them.
Topics: Animals; Biological Assay; Cell- and Tissue-Based Therapy; Humans; Quality Control
PubMed: 32589107
DOI: 10.2217/rme-2020-0003 -
Clinical & Translational Oncology :... Sep 2014The objective of this review is to summarize recent scientific and medical literature regarding chemoresponse assays or chemotherapy sensitivity and resistance assays... (Review)
Review
The objective of this review is to summarize recent scientific and medical literature regarding chemoresponse assays or chemotherapy sensitivity and resistance assays (CSRAs), specifically as applied to epithelial ovarian cancer. A total of sixty-seven articles, identified through PubMed using the key words "in vitro chemoresponse assay," "chemo sensitivity resistance assay," "ATP," "HDRA," "EDR," "MiCK," and "ChemoFx," were reviewed. Recent publications on marker validation, including relevant clinical trial designs, were also included. Recent CSRA research and clinical studies are outlined in this review. Published findings demonstrate benefits regarding patient outcome with respect to recent CSRAs. Specifically, analytical and clinical validations, as well as clinical utility and economic benefit, of the most common clinically used CSRA in the United States support its use to aid in making effective, individualized clinical treatment selections for patients with ovarian cancer.
Topics: Biological Assay; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Drug Resistance, Neoplasm; Female; Humans; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Research Design
PubMed: 24986099
DOI: 10.1007/s12094-014-1192-8 -
BMC Bioinformatics Nov 2010In recent years, the number of High Throughput Screening (HTS) assays deposited in PubChem has grown quickly. As a result, the volume of both the structured information...
BACKGROUND
In recent years, the number of High Throughput Screening (HTS) assays deposited in PubChem has grown quickly. As a result, the volume of both the structured information (i.e. molecular structure, bioactivities) and the unstructured information (such as descriptions of bioassay experiments), has been increasing exponentially. As a result, it has become even more demanding and challenging to efficiently assemble the bioactivity data by mining the huge amount of information to identify and interpret the relationships among the diversified bioassay experiments. In this work, we propose a text-mining based approach for bioassay neighboring analysis from the unstructured text descriptions contained in the PubChem BioAssay database.
RESULTS
The neighboring analysis is achieved by evaluating the cosine scores of each bioassay pair and fraction of overlaps among the human-curated neighbors. Our results from the cosine score distribution analysis and assay neighbor clustering analysis on all PubChem bioassays suggest that strong correlations among the bioassays can be identified from their conceptual relevance. A comparison with other existing assay neighboring methods suggests that the text-mining based bioassay neighboring approach provides meaningful linkages among the PubChem bioassays, and complements the existing methods by identifying additional relationships among the bioassay entries.
CONCLUSIONS
The text-mining based bioassay neighboring analysis is efficient for correlating bioassays and studying different aspects of a biological process, which are otherwise difficult to achieve by existing neighboring procedures due to the lack of specific annotations and structured information. It is suggested that the text-mining based bioassay neighboring analysis can be used as a standalone or as a complementary tool for the PubChem bioassay neighboring process to enable efficient integration of assay results and generate hypotheses for the discovery of bioactivities of the tested reagents.
Topics: Biological Assay; Data Mining; Databases, Factual; High-Throughput Screening Assays
PubMed: 21059237
DOI: 10.1186/1471-2105-11-549 -
American Journal of Physiology. Renal... Nov 2018Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted... (Review)
Review
Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted for study of lower urinary tract physiology. The spontaneous void spot assay (VSA), a popular micturition assessment tool, involves placing a mouse in an enclosure lined by filter paper and quantifying the resulting urine spot pattern. The VSA has advantages of being inexpensive and noninvasive, but some investigators challenge its ability to distinguish lower urinary tract function from behavioral voiding. A consensus group of investigators who regularly use the VSA was established by the National Institutes of Health in 2015 to address the strengths and weaknesses of the assay, determine whether it can be standardized across laboratories, and determine whether it can be used as a surrogate for evaluating urinary function. Here we leverage experience from the consensus group to review the history of the VSA and its uses, summarize experiments to optimize assay design for urinary physiology assessment, and make best practice recommendations for performing the assay and analyzing its results.
Topics: Animals; Biological Assay; Disease Models, Animal; Mice; Reproducibility of Results; Time Factors; Urinary Bladder; Urination; Urination Disorders; Urodynamics
PubMed: 30156116
DOI: 10.1152/ajprenal.00350.2018 -
Parasites & Vectors Jul 2023Numerous bioassay methods have been used to test the efficacy of repellents for ticks, but the comparability of results across different methods has only been evaluated...
BACKGROUND
Numerous bioassay methods have been used to test the efficacy of repellents for ticks, but the comparability of results across different methods has only been evaluated in a single study. Of particular interest are comparisons between bioassays that use artificial containers (in vitro) with those conducted on a human subject (in vivo) for efficacy testing of new potential unregistered active ingredients, which most commonly use in vitro methods.
METHODS
We compared four different bioassay methods and evaluated three ingredients (DEET [N,N-Diethyl-meta-toluamide], peppermint oil and rosemary oil) and a negative control (ethanol) over a 6-h period. Two of the methods tested were in vivo bioassay methods in which the active ingredient was applied to human skin (finger and forearm bioassays), and the other two methods were in vitro methods using artificial containers (jar and petri dish bioassays). All four bioassays were conducted using Ixodes scapularis nymphs. We compared the results using nymphs from two different tick colonies that were derived from I. scapularis collected in the US states of Connecticut and Rhode Island (northern origin) and Oklahoma (southern origin), expecting that ticks of different origin would display differences in host-seeking behavior.
RESULTS
The results between bioassay methods did not differ significantly, even when comparing those that provide the stimulus of human skin with those that do not. We also found that tick colony source can impact the outcome of repellency bioassays due to differences in movement speed; behavioral differences were incorporated into the assay screening. DEET effectively repelled nymphs for the full 6-h duration of the study. Peppermint oil showed a similar repellent efficacy to DEET during the first hour, but it decreased sharply afterwards. Rosemary oil did not effectively repel nymphs across any of the time points.
CONCLUSIONS
The repellency results did not differ significantly between the four bioassay methods tested. The results also highlight the need to consider the geographic origin of ticks used in repellency bioassays in addition to species and life stage. Finally, our results indicate a limited repellent efficacy of the two essential oils tested, which highlights the need for further studies on the duration of repellency for similar botanically derived active ingredients and for evaluation of formulated products.
Topics: Humans; Animals; Ixodes; DEET; Biological Assay; Connecticut; Ethanol; Insect Repellents; Nymph
PubMed: 37430360
DOI: 10.1186/s13071-023-05845-7