Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed Rapid Detection in Food and Feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 µg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 µg/kg by ELISA which correlated to >10 µg/kg by LC-MS/MS.

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Background: There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.

Methods: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.

Results: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.

Conclusions: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

A Statistical Model for Shadowed Body Centric Communications Channels: Theory and Validation

This paper presents a new statistical signal reception model for shadowed body-centric communications channels. In this model, the potential clustering of multipath components is considered alongside the presence of elective dominant signal components. As typically occurs in body-centric communications channels, the dominant or line-of-sight (LOS) components are shadowed by body matter situated in the path trajectory. This situation may be further exacerbated due to physiological and biomechanical movements of the body. In the proposed model, the resultant dominant component which is formed by the phasor addition of these leading contributions is assumed to follow a lognormal distribution. A wide range of measured and simulated shadowed body-centric channels considering on-body, off-body and body-to-body communications are used to validate the model. During the course of the validation experiments, it was found that, even for environments devoid of multipath or specular reflections generated by the local surroundings, a noticeable resultant dominant component can still exist in body-centric channels where the user's body shadows the direct LOS signal path between the transmitter and the receiver.

Development and validation of Fluid Structure Interaction Methods for an Oscillating Wave Surge Converter

The term fatigue loads on the Oyster Oscillating Wave Surge Converter (OWSC) is used to describe hydrostatic loads due to water surface elevation with quasi-static changes of state. Therefore a procedure to implement hydrostatic pressure distributions into finite element analysis of the structure is desired. Currently available experimental methods enable one to measure time variant water surface elevation at discrete locations either on or around the body of the scale model during tank tests. This paper discusses the development of a finite element analysis procedure to implement time variant, spatially distributed hydrostatic pressure derived from discretely measured water surface elevation. The developed method can process differently resolved (temporal and spatial) input data and approximate the elevation over the flap faces with user defined properties. The structural loads, namely the forces and moments on the body can then be investigated by post processing the numerical results. This method offers the possibility to process surface elevation or hydrostatic pressure data from computational fluid dynamics simulations and can thus be seen as a first step to a fluid-structure interaction model.

Collating the knowledge base for core outcome set development: developing and appraising the search strategy for a systematic review

Background: The COMET (Core Outcome Measures in Effectiveness Trials) Initiative is developing a publicly accessible online resource to collate the knowledge base for core outcome set development (COS) and the applied work from different health conditions. Ensuring that the database is as comprehensive as possible and keeping it up to date are key to its value for users. This requires the development and application of an optimal, multi-faceted search strategy to identify relevant material. This paper describes the challenges of designing and implementing such a search, outlining the development of the search strategy for studies of COS development, and, in turn, the process for establishing a database of COS.

Methods: We investigated the performance characteristics of this strategy including sensitivity, precision and numbers needed to read. We compared the contribution of databases towards identifying included studies to identify the best combination of methods to retrieve all included studies.

Results: Recall of the search strategies ranged from 4% to 87%, and precision from 0.77% to 1.13%. MEDLINE performed best in terms of recall, retrieving 216 (87%) of the 250 included records, followed by Scopus (44%). The Cochrane Methodology Register found just 4% of the included records. MEDLINE was also the database with the highest precision. The number needed to read varied between 89 (MEDLINE) and 130 (SCOPUS).

Conclusions: We found that two databases and hand searching were required to locate all of the studies in this review. MEDLINE alone retrieved 87% of the included studies, but actually 97% of the included studies were indexed on MEDLINE. The Cochrane Methodology Register did not contribute any records that were not found in the other databases, and will not be included in our future searches to identify studies developing COS. SCOPUS had the lowest precision rate (0.77) and highest number needed to read (130). In future COMET searches for COS a balance needs to be struck between the work involved in screening large numbers of records, the frequency of the searching and the likelihood that eligible studies will be identified by means other than the database searches.

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.