Tomato ionomic approach for food fortification and safety.

Food fortification is an issue of paramount of importance for people living both in developed and in developing countries. Among substances listed as "nutriceuticals", essential minerals have been recognised for their involvement in several healthy issues, involving all ages. In this frame, food plants are playing a pivotal role since their capability to compartmentalise ions and proteinmetal complexes in edible organs. Conversely, the accumulation of high metal levels in those organs may lead to safety problems. In the recent years, thanks to the availability of new and improved analytical apparatus in both ionic and genomic/transcrittomics areas, it is became feasible to couple data coming from plant physiology and genetics. Ionomics is the discipline that studies the cross-analysis of both data sets. Our group, in the frame of GenoPom project granted by MiUR, is interested to study the ionomics of tomatoes cultivars derived by breeding programmes in which wild relatives have been used to transfer several useful traits, such as resistance to biotic or abiotic stresses, fruit composition and textiture, etc. The introgression of the wild genome into the cultivated one produces new gene combinations. They might lead to the expression of some traits, such as increased or reduced adsorption of some metals and their exclusion or loading into edible organs, thus strongly involving the nutritional food value. Our final goal is to put together data coming from ions homeostasis and gene expression analyses, thus obtaining an ionomic tomato map related to ions absorption, translocation and accumulation in various plant organs, fruits included. To follow our hypothesis, we are studying the ionome of Solanum lycopersicum cv. M82 along with 76 Introgression Lines (ILs) produced by interspecific crosses between this cultivar and the wild species S. pennellii. These ILs are homozygous for small portions of the wild species genome introgressed into the domesticated M82 one. They are used as a useful tool for mapping QTL associated with many traits of interest. It is worthy to note that, until now, little information is available on QTL for ions accumulation in tomato. Moreover, as our knowledge, effects of new gene combinations in introgressed lines on ions uptake related to food safety have not been extensively studied. In this presentation we show results coming from the ionome analysis, carried out on S . lycopersicum M82 and several ILs. Plants were grown in pots in a greenhouse and watered with deionised water Thirty day-old plants were left to grow for 15 days in the presence of non-toxic concentration of Cd, Pb, As, Cr and Zn given combined. Leaves of all plants were then harvested and stored at -80°C for ionome and gene expression analyses. Preliminary results of ionome analysis of S. lycopersicum M82 and several ILs, carried out using an ICP-MS, showed that traits correlated to toxic metals and micronutrients accumulation in apical leaves were significantly modified in response to specific genetic backgrounds. Those results are perhaps due to the introgression of traits linked to uptake, translocation and accumulation of useful and/or toxic metal into plant apical leaves and to interactions of the wild type introgressed genomic regions with the cultivated genome. Also, data are shown on the identification and isolation of Solanum gene sequences related to ions uptake, translocation and accumulation, useful for further real-time gene expression evaluation in both cultivated and ILs during the treatments with the above-mentioned metals

Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord.

By light microscopic immunocytochemistry it has been previously shown that approximately equal to 70% of the neurons in rat dorsal root ganglia are labeled with an antiserum for glutamate conjugated to hemocyanin; the smaller among these neurons are also positive for substance P. By using a postembedding ImmunoGold method and electron microscopy, it is shown here that synaptic terminals in the superficial laminae of the spinal cord of rats selectively stain for the same glutamate antiserum. Immunolabeling is in small dome-shaped and in large scalloped synaptic terminals. Scalloped terminals are of two types. One type consists of dark terminals with many agranular vesicles of different size and a few large granular vesicles; these are probably endings of unmyelinated and small myelinated primary afferent fibers. The other type consists of light terminals with small agranular vesicles homogeneous in size with neurofilaments and many mitochondria; these are probably endings of larger myelinated primary afferent fibers. By means of double-labeling electron microscopic immunocytochemistry with colloidal gold particles of two different sizes, it is also shown here that substance P is present in only the dark type of glutamate-labeled scalloped terminals. The primary afferent origin of the terminals labeled by the antisera for glutamate and for substance P is demonstrated by a triple-labeling strategy: immunocytochemistry for both antisera on sections from rats in which dorsal rhizotomy or dorsal root ganglion injection of horseradish peroxidase conjugated to wheat germ agglutinin was performed. It is proposed that glutamate is the neurotransmitter in primary afferents mediating input from different peripheral receptor classes, including nociceptors. Effects of glutamate and substance P on spinal dorsal horn neurons may result from co-release of these two mediators from the same dorsal root afferent terminal

Approach combining the Rietveld method and pairs distribution function analysis to study crystalline materials under high-pressure and/or temperature: Application to rhombohedral Bi2Te3 phase

An approach combining the Rietveld method and pairs distribution function analysis to study crystalline materials under high pressure or temperature was early proposed by us, and in this study, it was applied to investigate de effect of high pressure on the rhombohedral Bi2Te3 phase. The refined structural parameters obtained from the Rietveld refinement of the XRD patterns measured for pressures up to 9.1 GPa were used as input data to simulate the partial and total structure factors SBiBi, SBiTe, STeTe, and SBi2Te3. Fourier transformation of the Sij factors permitted to obtain the partial and total pairs distribution functions GBiBi, GBiTe, GTeTe, and GBi2Te3. The first coordination shells of these Gij functions are formed by subshells and, with increasing pressure in the 1.1 to 6.3 GPa range, occur a partial separation of subshells. Also, the increase of pressure in this range promotes a drastic reduction in the values of the intralayer angles TeBiTe, and consequently, in the intralayer distance TeTe. A drastic reduction in the interlayers distance Te-Te was also observed. Several studies are reported in the literature, including one carried out by us, show the presence of an ETT in this pressure range. The obtained results suggest that the ETT is related with the decrease of the intralayer angles TeBiTe, and intra- and interlayer distance TeTe. Experimental results describing the pressure dependence the thermoelectric power, electrical resistivity, and power fator for rhombohedral Bi2Te3 are reported, and an enhancement of the power factor in the 1.1 to 6.3 GPa range is observed. The results obtained in this study give evidence that this enhancement in the power factor is related with the decrease of the intralayer angles TeBiTe, and with the decrease of intralayer- and interlayers homopolar TeTe bonds

Synthesis and anticancer activity of CDDO and CDDO-me, two derivatives of natural triterpenoids

Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me

DNA Topoisomerase I differentially modulates R-loops across the human genome

Background: Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown. Results: Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay. Conclusions: Our findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation

Ion uptake and YSL1 gene identification in tomato

Tomato breeder are using wild tomato relatives, even non-cross compatibles ones, in order to obtain cultivars with highly commercial values bearing new traits. However, the introgression of a wild genome into the cultivated one produces a new gene combinations that may lead to the expression of undeliverable traits, perhaps not so easy to recognise; even more, phenotypic variations may escape during the selection procedure when minor genes or non-abnormal phenotypes are involved. In the frame of the “GenoPom” project funded by MIUR, we have focused our interest on the alteration of heavy metals uptake from the soil and their loading into edible organs in commercial lines coming from Solanum interspecific crosses. Our final aim is to put together data coming from ion homeostasis and gene expression analyses, thus obtaining a ionomic map of tomato. To pursue our goal, we have started to study the cv M82 of Solanum lycopersicon, the wild relative Solanum pennelli and their introgression lines IL. Regarding the experiments on ion homeostasis, S. lycopersicon M82 and the introgression line IL 6-4-2 were grown in hydroponics under controlled environmental conditions. Twenty day-old plants were left to grow for 10 days in the presence of non-toxic concentration of Cd (10 mM), Pb (3 mM), Zn (100 mM) given separately or combined. Control and treated roots and leaves were then harvested and stored at -80°C for ionic and gene expression analyses. Ions analysis of Solanum lycopersicon M82 and IL 6-4-2 showed that traits correlated to ionic homeostasis is significantly modified in response to all metals and to the genotype. The analysis of ions data, obtained by ICP-MS, give a pictures of the different responses performed both to different stress and to combined stress, probably correlated to the up-regulation and/or down regulation of metal uptake proteins. Performed experiments demonstrate that the introgression of the wild genome into the cultivated one produces a new phenotype, perhaps due to the expression of traits linked to uptake, translocation and accumulation of useful and/or toxic metal into plant tissues and organs. Regarding the functional genomics approach for gaining insight into gene networks involved in mineral-ion accumulation in tomato plants, in literature has been reported that at least 25 major family genes are involved for metal homeostasis in plants. Among them, the genes ysl, hma, mtp, znt, zrt have been already studied at least in the plant species Arabidopsis thaliana, A. halleri and Thlaspi caerulescens. So far, no such genes have been reported to be cloned in Solanum species. We have focused our study on the genes YSL1, ZNT1 and MTP1 responsible for uptake, translocation and accumulation of metal such as zinc, cadmium, and iron into plant compartment. For all of them, consensous sequences from nucleotide multialignment have been obtained. Then, each of those were blasted in a Solanum EST collection databank and an assembled UniGene sequence was obtained.. Finally, we have designed primers and performed PCR analysis on S. lycopersicon and S. pennelli genomic DNA. So far, we have cloned a putative ysl1 sequence from tomato, that has shown that a very high percentage of identity (92%) with whole ysl1 gene of Nicotiana tabacum; the in silico translated sequence of this sequence has shown a 89% of identity with the same tobacco protein