Browsing by Author "Carter, David"

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    Discrimination between aflatoxigenic and non-aflatoxigenic Aspergillus section Flavi strains from Egyptian peanuts using molecular and analytical techniques
    (Wageningen Academic Publishers, 2011-02-01T00:00:00Z) Abdel-Hadi, Ahmed; Carter, David; Magan, Naresh
    A wide range of Aspergillus section Flavi strains were isolated from Egyptian peanut samples. Eighteen of these strains were compared with two type strains (Aspergillus flavus SRRC G1907 and Aspergillus parasiticus 2747) for aflatoxin production based on (a) qualitative fluorescence using a coconut cream agar medium (CAM), and (b) aflatoxin production on a conducive Yeast Extract-Sucrose (YES) medium using HPLC. These results were validated by using molecular approaches (the structural genes, aflD (nor-1), aflM (ver-1) and aflP (omt A) and the regulatory gene aflR) to discriminate between aflatoxigenic and non- aflatoxigenic strains of the Aspergillus section Flavi group in vitro and on peanut seeds. Overall, 13/18 strains producing aflatoxins B1 and B2 in the range 1.27-213.35 µg/g medium were identified. In addition, 5 non-aflatoxin producing strains were found. The expression of these four genes was assessed using PCR and RT-PCR. PCR showed that all strains contained the four aflatoxin genes examined, regardless of expression profiles. Our results also showed that aflD expression is a reliable marker to discriminate between aflatoxin and non- aflatoxin producers. Interestingly, when an aflatoxin producing strain and three non-aflatoxigenic strains were subsequently grown on peanuts at 0.95 water activity, two of the non-producers were able to initiate aflatoxin biosynthesis. This suggests that growth of strains on the natural food matrix is important for confirming aflatoxigenic production potential
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    Molecular ecology of aspergillus section flavi species : approaches to understand the role of aflatoxin genes in aflatoxin biosynthesis
    (Cranfield University, 2011-02) Abdel-Hadi, Ahmed; Magan, Naresh; Carter, David
    This is the first study to integrate and correlate the effect of ecophysiological factors on the life cycle of Aspergillus flavus by carrying out complementary work on gene expression of the aflatoxin gene cluster, with growth, sporulation and phenotypic toxin production. This information was used to understand the role of ecological factors on key biosynthetic genes and examine the use of such information for control of aflatoxin production using RNA interference. Ecological studies showed the profiles for growth, sporulation and aflatoxin B1 (AFB1) production with optimum ranges of water activity (aw) and temperature for AFB1 production being identified. A. flavus grew faster at 0.99 aw at all temperatures, but optimally at 30-35°C. The highest amount of asexual conidia was produced at 0.95 aw followed by 0.90 aw and then 0.99 aw at all temperatures examined. Interestingly, the partitioning of AFB1 into biomass, medium and spores showed that at 0.99 aw, about 50% of the mycotoxin was present in the biomass and the medium, with very little present in the spores. However, as water stress was imposed there was a switch to a significantly higher channelling of AFB1 (about 45%) into the spores, especially at 0.95 and 0.93 aw levels. A microarray analysis was used to examine the effect of aw x temperature interactions on the relative expression of the aflatoxin gene cluster for the first time using A. flavus NRRL 3357. This showed that under mild stress conditions (20°C/0.99 aw) several of the cluster genes, in particular aflS and aflJ, were highly induced concomitant with high levels of phenotypic AFB1 production. Highest amounts of AFB1 were produced in all conditions where aflS expression was elevated. When the ratio between the normalised expression data of the aflS/aflR genes was generated, high ratios were obtained at 25°C and 30°C at 0.99 and 0.95 aw and low ratios at 25°C and 30°C at 0.90 aw. This is in agreement with the AFB1 production profile. Cont/d.
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    The role of non-coding RNAs in haemoglobin regulation
    (Cranfield University, 2009) Trujillano Lidon, Daniel; Carter, David
    Non-coding RNAs appear to play a role in gene regulation by modulating chromatin structure. There is mounting evidence suggesting an essential role for non-coding RNAs in the complex process of the genetic regulation of the β-globin locus. Preliminary observations indicate that the BGL3 non-coding transcript may be involved in an RNA-protein interaction and may be interacting with chromatin in the β-globin locus as part of a regulatory function within the locus. However, the expression profile of this non-coding transcript has not yet been characterized and nothing is known about its mode of action. Here it is shown that the BGL3 transcript is dynamically up-regulated upon haemin induction of the K562 cell line (a human erythroleukemic cell line). To determine whether there is a correlation between the BGL3 transcript expression and the expression of the γ- and β-globin genes, the levels of the BGL3 transcript in K562 cells were perturbed by knocking it down using the RNA interference pathway. The effect of the knockdown of the BGL3 transcript was tested on the expression levels of the γ- and β-globin genes, which were quantified using qRT-PCR. Our results are the first, to our knowledge, that describe a developmentally regulated expression of the BGL3 non-coding transcript in haemin-induced K562 cells, and provide evidence that suggests that this transcript may be involved in the silencing of the β-globin gene.
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    Water quality from contrasting drained clay soils : the relative importance of sorbed and aqueous phase transport mechanisms
    (Cranfield University, 1997) Hardy, I. A. J.; Carter, David; Leeds-Harrison, Peter B.
    A full understanding of the processes controlling the movement of pesticides within the soil 1 water regime is critical to the successful reduction of losses of these substances to surface waters. Until recent years the importance of absorbed phase transport of pesticides has largely been ignored with respect to drainflow and the appearance of strongly absorbed hydrophobic compounds in riverine sediments has hitherto been largely attributed to surface runoff 1 erosion. Field studies were setup for the 1994/5 winter season to investigate the hydrology and chemistry associated with drainflow from the lighter clay soils of Boarded Barns Farm, with two calcareous (Hanslope and Stretham) and two non-calcareous (Melford and Ludford) soils being chosen for study. Instrumented study plot areas were established in each field, with drainflow measurement and water sampling being automated. Laboratory based investigations (including rainfall simulation and desorption) were carried out to support, extend and clarify the field data, in particular the processes involved in the transport of isoproturon and sediment. A striking feature seen in the field studies was the appearance of large amounts of suspended sediment in drainflow from the Melford soil, in contrast to the clear drainflow from the calcareous soils. The sediments in drain flow were identified as being of surface origin, generated by rainfall impact and transported via preferential by-pass flow. Laboratory investigations have indicated that the chemical nature of the topsoil is a dominant factor controlling the transport of sediments and clay flocculation due to the presence of calcium carbonate is proposed as a mechanism for preventing sediment transport in the calcareous soils. Where sediment was found in drainflow, it was shown to be responsible for the transport and losses in the absorbed phase of not only diflufenican, a strongly absorbed pesticide, but also isoproturon, a weakly absorbed pesticide. The laboratory studies indicate that isoproturon is transported predominantly absorbed to sediment and later desorbs 1 diffuses slowly into the aqueous phase of the receiving waters. The results of the field experiments were confirmed by the rainfall simulation studies on 20cm deep (20cm diameter) topsoil cores taken from the field, which showed sediment transport only in the non-calcareous soil. The extent of this phenomena of sediment transport in the UK is not well defined, due to the historical lack of research, however soils known to show the effect have a land coverage of 6% of England and Wales. The present study highlights an additional mechanism by which pesticide losses to surface waters may occur, but the relative importance will depend heavily on the hydrological, structural and chemical nature of the soil system.