Transcriptomic characterization of two major Fusarium resistance quantitative trait loci (QTLs), Fhb1 and Qfhs.ifa-5A, identifies novel candidate genes

Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248

Simultaneous determination of Deoxynivalenol, Deoxynivalenol-3-Glucoside and Nivalenol in wheat grains by HPLC-PDA with immunoaffinity column cleanup

Deoxynivalenol-3-glucoside (D3G) is a modified mycotoxin formed by the metabolism of plants through the conjugation of deoxynivalenol (DON) with glucose. Toxicology studies of D3G for human and animal health are still under investigation, and the development of practical and reliable methods for its direct determination, especially in cereal matrices, is of great importance. In the present study, a methodology for simultaneous determination of D3G, DON, and nivalenol (NIV) in wheat grains, using immunoaffinity column (IAC) cleanup, separation by C18 column and detection by ultraviolet (UV) absorption, was optimized and in-house validated. The results demonstrated adequate values of D3G recovery from IAC and spiked samples. Intraday precision, linearity, limit of detection and limit of quantification (LOQ) were also adequate for the determination of these mycotoxins. Range of applicability varied from 47.1 to 1000 g/kg for D3G and from 31.3 to 1000 g/kg for DON and NIV, with recovery ranging from 84.7±7.2 % to 112.3±8.1Felipe Trombete is grateful for a doctoral fellowship provided by the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES)

Low frequency of cigarette smoking and the risk of head and neck cancer in the INHANCE consortium pooled analysis.

Abstract BACKGROUND: Cigarette smoking is a major risk factor for head and neck cancer (HNC). To our knowledge, low cigarette smoking (<10 cigarettes per day) has not been extensively investigated in fine categories or among never alcohol drinkers. METHODS: We conducted a pooled analysis of individual participant data from 23 independent case-control studies including 19 660 HNC cases and 25 566 controls. After exclusion of subjects using other tobacco products including cigars, pipes, snuffed or chewed tobacco and straw cigarettes (tobacco product used in Brazil), as well as subjects smoking more than 10 cigarettes per day, 4093 HNC cases and 13 416 controls were included in the analysis. The lifetime average frequency of cigarette consumption was categorized as follows: never cigarette users, >0-3, >3-5, >5-10 cigarettes per day. RESULTS: Smoking >0-3 cigarettes per day was associated with a 50% increased risk of HNC in the study population [odds ratio (OR) = 1.52, 95% confidence interval (CI): (1.21, 1.90). Smoking >3-5 cigarettes per day was associated in each subgroup from OR = 2.01 (95% CI: 1.22, 3.31) among never alcohol drinkers to OR = 2.74 (95% CI: 2.01, 3.74) among women and in each cancer site, particularly laryngeal cancer (OR = 3.48, 95% CI: 2.40, 5.05). However, the observed increased risk of HNC for low smoking frequency was not found among smokers with smoking duration shorter than 20 years. CONCLUSION: Our results suggest a public health message that low frequency of cigarette consumption contributes to the development of HNC. However, smoking duration seems to play at least an equal or a stronger role in the development of HNC

Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography–tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100

Safe food and feed through an integrated toolbox for mycotoxin management: the MyToolBox approach

There is a pressing need to mobilise the wealth of knowledge from the international mycotoxin research conducted over the past 25-30 years, and to perform cutting-edge research where knowledge gaps still exist. This knowledge needs to be integrated into affordable and practical tools for farmers and food processors along the chain in order to reduce the risk of mycotoxin contamination of crops, feed and food. This is the mission of MyToolBox – a four-year project which has received funding from the European Commission. It mobilises a multi-actor partnership (academia, farmers, technology small and medium sized enterprises, food industry and policy stakeholders) to develop novel interventions aimed at achieving a significant reduction in crop losses due to mycotoxin contamination. Besides a field-to-fork approach, MyToolBox also considers safe use options of contaminated batches, such as the efficient production of biofuels. Compared to previous efforts of mycotoxin reduction strategies, the distinguishing feature of MyToolBox is to provide the recommended measures to the end users along the food and feed chain in a web-based MyToolBox platform (e-toolbox). The project focuses on small grain cereals, maize, peanuts and dried figs, applicable to agricultural conditions in the EU and China. Crop losses using existing practices are being compared with crop losses after novel pre-harvest interventions including investigation of genetic resistance to fungal infection, cultural control (e.g. minimum tillage or crop debris treatment), the use of novel biopesticides suitable for organic farming, competitive biocontrol treatment and development of novel modelling approaches to predict mycotoxin contamination. Research into post-harvest measures includes real-time monitoring during storage, innovative sorting of crops using vision-technology, novel milling technology and studying the effects of baking on mycotoxins at an industrial scale

Patterns of urgent hoarseness referrals to ENT – When should we be suspicious of cancer?

Background: Current UK referral criteria stipulate that hoarseness should be persistent to merit 2 week wait (2WW) or urgent suspicion of cancer (USOC) referral. This study delineates patterns of hoarseness presentation with a view to assisting referral pathways, and whereby reassurance could be provided. Methods: A pre-existing database of patients referred with hoarseness under the urgent suspicion of cancer (USOC) category was analysed. Univariate and multivariate analyses were performed on a variety of demographic and comorbid features to produce odds ratios (OR) of features either related or not related to laryngeal cancer. Results: Of 698 consecutive hoarseness referrals were studied. In these referrals there were 506(73%) with persistent hoarseness and 192(27%) with intermittent hoarseness. The most significant patient variables related to laryngeal cancer were persistent hoarseness (OR 4.97), recreational drug use (OR 4.94), male gender (OR 4.01) and weight loss (OR 3.75). Significant patient variables present not related to laryngeal cancer diagnosis were intermittent hoarseness (OR 0.2), the presence of cough (OR 0.2), globus sensation (OR 0.25) and recent viral infection (OR 0.29). Conclusion: The strongest association with cancer is seen in patients that are persistently hoarse. Patients with fluctuating hoarseness do not need an “urgent suspicion of cancer” referral. Additional demographic referral information could help to streamline the referral of these patients, and reassure others

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection - DAD and mass spectrometry - MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction - molecularly imprinted solid-phase extraction - liquid chromatography) produced high (R ≈ 95–98%) and repeatable (RSD < 3%) recovery values for ZON and α-ZOL