The application of Reporter Gene Assays for the detection of endocrine disruptors in sport supplements.

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC50 of 0.01 ng mL-1 and 0.16 ng mL-1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC–MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC–MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.

Neural network forecasts of input-output technology

A significant part of the literature on input-output (IO) analysis is dedicated to the development and application of methodologies forecasting and updating technology coefficients and multipliers. Prominent among such techniques is the RAS method, while more information demanding econometric methods, as well as other less promising ones, have been proposed. However, there has been little interest expressed in the use of more modern and often more innovative methods, such as neural networks in IO analysis in general. This study constructs, proposes and applies a Backpropagation Neural Network (BPN) with the purpose of forecasting IO technology coefficients and subsequently multipliers. The RAS method is also applied on the same set of UK IO tables, and the discussion of results of both methods is accompanied by a comparative analysis. The results show that the BPN offers a valid alternative way of IO technology forecasting and many forecasts were more accurate using this method. Overall, however, the RAS method outperformed the BPN but the difference is rather small to be systematic and there are further ways to improve the performance of the BPN.

Conservation genetics of Neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2-11 polymorphic microsatellite loci revealed only 5 diploid males at an overall frequency of 0.005 (95% CIs 0.002-0.010); errors through genetic non-detection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

Electron attachment to 2-nitro-m-xylene

Electron attachment to nitroaromatic compound 2-nitro-m-xylene in gas phase has been performed utilizing a double focusing two sector mass spectrometer with high mass resolution (m/Delta m approximate to 2500). At low energy (below 20 eV), electron interactions with the neutral 2-nitro-m-xylene molecule reveal a very rich fragmentation pattern. A total of 60 fragment anions have been detected and the ion yield for all observed negative ions has been recorded as a function of the incident electron energy, among them a long lived (metastable) non-dissociated parent anion which is formed at energies near zero eV, and some ions observed at the mass numbers 26,42 and 121. Comparison of calculated isotopic patterns with measured ion yields for these fragment anions and their successors in the mass spectrum, allows the assignment of the chemical composition of these fragments as CN- (26 Da), CNO- (42 Da) and C8H9O- (121 Da). Electron attachment to 2-nitro-m-xylene leads to anion formation at four energy ranges. Between 0 eV and 2 eV only few product ions are formed. Between 4.6 eV and 6.1 eV all fragment anions are formed and for most of them the anion yield reaches its maximum value in this range. NO2- which is the most abundant product [M-H](-) and O- are the only fragments that exhibit a feature at 7.4eV, 8.1 eV and 7.9eV, respectively. About half of the fragment anions exhibit a broad, mostly low-intensity resonance between 9 eV and 10 eV. (C) 2009 Elsevier B.V. All rights reserved.

Time of flight mass spectrometry for quantitative data analysis in fast transient studies using a Temporal Analysis of Products (TAP) reactor

A Time of flight (ToF) mass spectrometer suitable in terms of sensitivity, detector response and time resolution, for application in fast transient Temporal Analysis of Products (TAP) kinetic catalyst characterization is reported. Technical difficulties associated with such application as well as the solutions implemented in terms of adaptations of the ToF apparatus are discussed. The performance of the ToF was validated and the full linearity of the specific detector over the full dynamic range was explored in order to ensure its applicability for the TAP application. The reported TAP-ToF setup is the first system that achieves the high level of sensitivity allowing monitoring of the full 0-200 AMU range simultaneously with sub-millisecond time resolution. In this new setup, the high sensitivity allows the use of low intensity pulses ensuring that transport through the reactor occurs in the Knudsen diffusion regime and that the data can, therefore, be fully analysed using the reported theoretical TAP models and data processing.

Global down-regulation of HOX gene expression in PML-RAR alpha(+) acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex net-Work of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha, fusion proteins have been reported to act as part of a repressor complex during myelold cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Stir bar sorptive extraction of diclofenac from liquid formulations: A proof of concept study

A new stir bar sorptive extraction (SBSE) technique coupled with HPLC-UV method for quantification of diclofenac in pharmaceutical formulations has been developed and validated as a proof of concept study. Commercially available polydimethylsiloxane stir bars (Twister (TM)) were used for method development and SBSE extraction (pH, phase ratio, stirring speed, temperature, ionic strength and time) and liquid desorption (solvents, desorption method, stirring time etc) procedures were optimised. The method was validated as per ICH guidelines and was successfully applied for the estimation of diclofenac from three liquid formulations viz. Voltarol (R) Optha single dose eye drops, Voltarol (R) Ophtha multidose eye drops and Voltarol (R) ampoules. The developed method was found to be linear (r=0.9999) over 100-2000 ng/ml concentration range with acceptable accuracy and precision (tested over three QC concentrations). The SBSE extraction recovery of the diclofenac was found to be 70% and the LOD and LOQ of the validated method were found to be 16.06 and 48.68 ng/ml, respectively. Furthermore, a forced degradation study of a diclofenac formulation leading to the formation of structurally similar cyclic impurity (indolinone) was carried out. The developed extraction method showed comparable results to that of the reference method, i.e. method was capable of selectively extracting the indolinone and diclofenac from the liquid matrix. Data on inter and intra stir bar accuracy and precision further confirmed robustness of the method, supporting the multiple re-use of the stir bars. (C) 2010 Elsevier B.V. All rights reserved.

Developments in the production of biological and synthetic binders for immunoassay and sensor-based detection of small molecules

The need for chemical and biological entities of predetermined selectivity and affinity towards target analytes is greater than ever, in applications such as environmental monitoring, bioterrorism detection and analysis of natural toxin contaminants in the food chain.

Advances in biosensor-based analysis for antimicrobial residues in foods

Biosensors are used for a large number of applications within biotechnology, including the pharmaceutical industry and life sciences. Since the production of Biacore surface-plasmon resonance instruments in the early 1990s, there has been steadily growing use of this technology for the detection of food contaminants (e.g., veterinary drugs, mycotoxins, marine toxins, food dyes and processing contaminants). Other biosensing technologies (e.g., electrochemical and piezoelectric) have also been employed for the analysis of small-molecule contaminants. This review concentrates on recent advances made in detection and quantification of antimicrobial compounds with different types of biosensors and on the emergence of multiplexing, which is highly desirable as it increases sample analysis at lower cost and in less time. (C) 2010 Elsevier Ltd. All rights reserved.

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BC) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BC, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BC which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes. (C) 2011 Elsevier B.V. All rights reserved.

In vitro bioassays for the study of endocrine-disrupting food additives and contaminants

Endocrine-disrupting chemicals (EDCs) are capable of interfering with normal hormone homeostasis by acting on several targets and through a wide variety of mechanisms. Unwanted exposure to EDCs can lead to a wide spectrum of adverse health effects, especially when exposure is during critical windows of development. Feed and food are considered to be among the main routes of inadvertent exposure to EDCs, so there is an important need for efficient detection of EDCs in these matrices.