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Pleil J.D.,U.S. Environmental Protection Agency | Angrish M.M.,US Toxicology | Madden M.C.,Health-U
Journal of Breath Research | Year: 2015

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-, IL-1β and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r2 > 0.99). Sensitivities varied by analyte, but were robust from ∼0.006 (IL-6) to ∼0.01 (TNF-) pg ml-1. All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ∼45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml-1). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels. © 2015 IOP Publishing Ltd.

Simmons S.O.,US Toxicology
Combinatorial Chemistry and High Throughput Screening | Year: 2011

Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chemicals currently in commercial use. In addition, there are social and ethical pressures to reduce, refine and replace animal testing in toxicology. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chemical toxicants. Critical to this vision, assays that measure toxicity pathways associated with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biological activities in a quantitative and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assay formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays. © 2011 Bentham Science Publishers Ltd.

Llorens A.,CSIC - Institute of Agricultural Chemistry and Food Technology | Lloret E.,IRTA - Institute of Agricultural-Alimentary Research and Technology | Picouet P.A.,IRTA - Institute of Agricultural-Alimentary Research and Technology | Trbojevich R.,US Toxicology | Fernandez A.,CSIC - Institute of Agricultural Chemistry and Food Technology
Trends in Food Science and Technology | Year: 2012

Metallic-based micro and nano-structured materials are incorporated into food contact polymers to enhance mechanical and barrier properties, and to prevent the photodegradation of plastics. Additionally heavy metals are effective antimicrobials in the form of salts, oxides, and colloids, complexes such as silver zeolites, or as elemental nanoparticles. They are incorporated for food preservation purposes and to decontaminate surfaces in industrial environments. Other relevant properties in active food packaging, such as the capability for ethylene oxidation or oxygen scavenging, can be used to extend food shelf-life. Silver based nano-engineered materials are currently the most commonly used in commodities due to their antimicrobial capacity. Copper, zinc and titanium nanostructures are also showing promise in food safety and technology. The antimicrobial properties of zinc oxide at the nanoscale will provide affordable and safe innovative strategies. Copper has been shown to be an efficient sensor for humidity, and titanium oxide has resistance to abrasion and UV-blocking performance. The migration of cations from the polymer matrices is the key point to determine their antimicrobial effectiveness; however, this cation migration may affect legal status of the polymer as a food-contact material. © 2011 Elsevier Ltd.

Beger R.D.,National Center for Toxicological Research (NCTR) | Flynn T.J.,US Toxicology
Metabolomics | Year: 2016

Background: Pharmacometabolomics is a relatively new field that measures an individual’s metabolome in biofluids to detect prognostic and diagnostic biomarkers of drug response and to provide an effective means to predict variation in a subject’s response to drug treatment. Pharmacometabolomics has the potential to help clinicians determine the effectiveness and safety of a drug on an individual basis. Aim of Review: To provide information from the current literature in pharmocometabolomics relevant to drug safety including factors besides genetics that can play a role in how a subject responds to a drug treatment. Pharmacometabolomics studies on drug-induced liver toxicity, the use of pharmacometabolomics to detect and predict drug interactions, and future applications of pharmacometabolomics in drug safety are discussed. Key scientific concepts of the review: Pharmacometabolomics can play a role in identifying and/or characterizing toxicity at all stages of drug development. These stages include: pharmacokinetics and ADME; initial toxicity; protective mechanisms; adverse events; late injury; and, injury progression or recovery. Pharmacometabolomics also has the ability to detect endogenous metabolites and markers of other exposure factors including alcohol consumption, impact of the gut microbiome, nutrition, other medications (polypharmacy), dietary supplements, and current individual health-to-disease status, all of which could play a role in patient response to a drug. Pharmacometabolomics alone or in combination with pharmacogenomics can be used to develop customized treatment plans for patients (i.e., personalized medicine) that could significantly reduce adverse events that are sometimes associated with the use of pharmaceuticals. © 2016, Springer Science+Business Media New York (outside the USA).

Cheng Y.,Texas A&M University | Jutooru I.,Texas A&M University | Jutooru I.,Covance | Chadalapaka G.,Texas A&M University | And 2 more authors.
Oncotarget | Year: 2015

HOTTIP is a long non-coding RNA (lncRNA) transcribed from the 5′ tip of the HOXA locus and is associated with the polycomb repressor complex 2 (PRC2) and WD repeat containing protein 5 (WDR5)/mixed lineage leukemia 1 (MLL1) chromatin modifying complexes. HOTTIP is expressed in pancreatic cancer cell lines and knockdown of HOTTIP by RNA interference (siHOTTIP) in Panc1 pancreatic cancer cells decreased proliferation, induced apoptosis and decreased migration. In Panc1 cells transfected with siHOTTIP, there was a decrease in expression of 757 genes and increased expression of 514 genes, and a limited gene analysis indicated that HOTTIP regulation of genes is complex. For example, Aurora kinase A, an important regulator of cell growth, is coregulated by MLL and not WDR5 and, in contrast to previous studies in liver cancer cells, HOTTIP does not regulate HOXA13 but plays a role in regulation of several other HOX genes including HOXA10, HOXB2, HOXA11, HOXA9 and HOXA1. Although HOTTIP and the HOX-associated lncRNA HOTAIR have similar pro-oncogenic functions, they regulate strikingly different sets of genes in Panc1 cells and in pancreatic tumors.

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