Emanuela Azara

Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum, the incitant of crown and foot rot on wheat. The transformation of a wild-type strain and its nitrate reductase-deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcription-polymerase chain reaction, corresponding to a TRI6-hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28-fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild-type.

Simultaneous amperometric detection of ascorbic acid and antioxidant capacity in orange, blueberry and kiwi juice, by a telemetric system coupled with a fullerene- or nanotubes-modified ascorbate subtractive biosensor

Four fullerenes- or nanotubes-modified graphite sensor-biosensor systems (SBs), coupled with a dual-channel telemetric device, based on an ascorbate oxidase (AOx) biosensor, were developed for on line simultaneous amperometric detection of ascorbic acid (AA) and antioxidant capacity in blueberry, kiwi and orange juice. Fullerene C60 (FC60), fullerene C70 (FC70), single-walled carbon nanotubes (SWCN) and multi-walled carbon nanotubes (MWCN) increased the sensitivity of graphite toward AA and phenols 1.2, 1.5, 5.1 and 5.1 times respectively. Fullerenes combined with AOx improved the selectivity toward AA more than nanotubes, being able to hold a higher number of AOx molecules on the biosensor surface. The SBs work at an applied potential of +500 mV, in a concentration range between the LOD and 20 μM, with a response time of two minutes. The LOD is 0.10, 0.13, 0.20 and 0.22 μM for SBs modified with FC60, FC70, SWCN and MWCN respectively. Biosensors register lower AA currents than the sensors due to the enzyme capability to oxidize AA before it reaches the transductor surface. Phenols currents registered by sensors and biosensors did not differ. Based on the difference between sensor and biosensor recorded currents a AA selectivity index was developed as an indicator of specificity toward AA and of the capacity to distinguish between AA and phenols contribution to the antioxidant capacity. This value is almost zero for fullerene-modified SBs, 0.13 and 0.22 for SWCN- and MWCN-modified SBs respectively. The results of juices analysis performed with SBs were in accordance with reference methods.

Natural and Natural-like Phenolic Inhibitors of Type B Trichothecene in Vitro Production by the Wheat (Triticum sp.) Pathogen Fusarium culmorum

Fusarium culmorum, a fungal pathogen of small grain cereals, produces 4-deoxynivalenol and its acetylated derivatives that may cause toxicoses on humans or animals consuming contaminated food or feed. Natural and natural-like compounds belonging to phenol and hydroxylated biphenyl structural classes were tested in vitro to determine their activity on vegetative growth and trichothecene biosynthesis by F. culmorum. Most of the compounds tested at 1.5 or 1.0 mM reduced 3-acetyl-4-deoxynivalenol production by over 70% compared to the control, without affecting fungal growth significantly. Furthermore, several compounds retained their ability to inhibit toxin in vitro production at the lowest concentrations of 0.5 and 0.25 mM. Magnolol 27 showed fungicidal activity even at 0.1 mM. No linear correlation was observed between antioxidant properties of the compounds and their ability to inhibit fungal growth and mycotoxigenic capacity. A guaiacyl unit in the structure may play a key role in trichothecene inhibition.

LC-MS analysis of trimethoxyamphetamine designer drugs (TMA series)from urine samples

A sensitive liquid chromatography-mass spectrometric (LC-MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C(18) cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 microm Polar Plus column (150 mm x 2.1 mm) with acetonitrile -0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10-200 ng/mL) (R(2) > or = 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70-10.77% and 7.63-12.94%, respectively. This LC-MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.

Molecular changes induced by the curcumin analogue D6 in human melanoma cells

BackgroundIn a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells.ResultsTo shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis.ConclusionsOur findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

Development and Characterization of an Ascorbate Oxidase-based Sensor–Biosensor System for Telemetric Detection of AA and Antioxidant Capacity in Fresh Orange Juice

A new carbon ascorbate oxidase-based sensor-biosensor system (SB) was coupled to a dual-channel telemetric device for online simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello, and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD 0.26 μM and 20 μM. In this concentration range, limiting the data acquisition time to 2 min, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods.

Natural Phenolic Inhibitors of Trichothecene Biosynthesis by the Wheat Fungal Pathogen Fusarium culmorum: A Computational Insight into the Structure-Activity Relationship

A model of the trichodiene synthase (TRI5) of the wheat fungal pathogen and type-B trichothecene producer Fusarium culmorum was developed based on homology modelling with the crystallized protein of F. sporotrichioides. Eight phenolic molecules, namely ferulic acid 1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, eugenol dimer 6, magnolol 7, and ellagic acid 8, were selected for their ability to inhibit trichothecene production and/or fungal vegetative growth in F. culmorum. The chemical structures of phenols were constructed and partially optimised based on Molecular Mechanics (MM) studies and energy minimisation by Density Functional Theory (DFT). Docking analysis of the phenolic molecules was run on the 3D model of F. culmorum TRI5. Experimental biological activity, molecular descriptors and interacting-structures obtained from computational analysis were compared. Besides the catalytic domain, three privileged sites in the interaction with the inhibitory molecules were identified on the protein surface. The TRI5-ligand interactions highlighted in this study represent a powerful tool to the identification of new Fusarium-targeted molecules with potential as trichothecene inhibitors.

Abstract 3804: Molecular changes induced by the curcumin biphenyl analogue D6 in melanoma cells

Malignant melanoma (MM) is one of the most aggressive cancers that affect humans. It is the most common and fatal skin cancer, with an incidence that is critically increasing in Western populations (15 times in the last 40 years) and for which, in the advanced stages, there is no effective therapy. The search for new compounds able to control it is therefore essential. The hydroxylated biphenyl compound (3E,3′E)-4,4′-(5,5′,6,6′-tetramethoxy-[1,1′-biphenyl]-3,3′-diyl)bis(but-3-en-2-one), namely D6, a structural analogue of curcumin, showed a strong antitumor activity on MM cells both in vitro and in vivo, as we have recently described [1]. The mechanisms of action of this compound are not clear yet, but it inhibits cancer cells growth by inducing apoptosis without affecting normal fibroblasts growth. Firstly we investigated the ability of D6 to enter cells by Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. The LC-MS data showed a quick cellular uptake of D6 with a maximum peak after about 2 hours. To investigate on the effects of D6 treatment on the cell cycle progression in MM cells, we performed flow cytometry analysis, which evidenced a cell cycle arrest in the G2/M phase. Gene expression profiles analysis using Illumina® high-density microarrays, was carried out on D6 treated MM cells and normal fibroblasts allowing the identification of 599 transcripts whose expression was modulated by D6 treatment in MM cells but not in normal fibroblast. The analysis of results, performed by Ingenuity Pathway Analysis software, showed the centrality of CDKN1A gene, encoding p21, a major negative cell cycle regulator, which was strongly over expressed, while cyclin B and cdc25 phosphatase, showed both to be down modulated. Other genes which expression interestingly appeared to be decreased by D6 activity were the oncogenes c-kit and PI3K, suggesting a down regulation of both c-kit mediated and PI3K/AKT signal transduction pathways driving to cell proliferation. Modulation of expression of these specific genes has been confirmed at a protein level by western blotting analysis. Moreover our results pointed out an upregulation of several heat shock family genes, which is probably due to a curcumin related anti inflammatory effect of D6. The preliminary results of these analysis show that D6 induces molecular changes in MM cells at both gene expression and protein levels, involving major cell proliferation regulatory pathways. [1] Marina Pisano et al. (2010).Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells. Molecular Cancer, 9:137. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3804. doi:1538-7445.AM2012-3804