Rina Venerando

Ternary Hybrid γ‐Fe2O3/CrVI/Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue

Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts.

Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern

RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.

Redox status in a model of cancer stem cells.

Reversible oxidation of Cys residues is a crucial element of redox homeostasis and signaling. According to a popular concept in oxidative stress signaling, the oxidation of targets of signals can only take place following an overwhelming of the cellular antioxidant capacity. This concept, however, ignores the activation of feedback mechanisms possibly leading to a paradoxical effect. In a model of cancer stem cells (CSC), stably overexpressing the TAZ oncogene, we observed that the increased formation of oxidants is associated with a globally more reduced state of proteins. Redox proteomics revealed that several proteins, capable of undergoing reversible redox transitions, are indeed more reduced while just few are more oxidized. Among the proteins more oxidized, G6PDH emerges as both more expressed and activated by oxidation. This accounts for the observed more reduced state of the NADPH/NADP+ couple. The dynamic redox flux generating this apparently paradoxical effect is rationalized in a computational system biology model highlighting the crucial role of G6PDH activity on the rate of redox transitions eventually leading to the reduction of reversible redox switches.

Mechanism of Autophagy in Permeabilized Hepatocytes

Autophagic vacuoles in hepatocytes are formed from membranes of rough and smooth endoplasmic reticulum (ER) by processes that are under immediate physiologic control by amino acids, insulin, and glucagon (reviewed in 1). Little, though, is known of the molecular steps involved. Microinjection (2) and electropermeabilization (3) have been used to introduce markers into cells and newly formed vacuoles. But because the pores are transient, observations are restricted to events that occur after membrane resealing. In order to gain access to autophagy under steady state conditions, we permeabilized hepatocytes with α-toxin from Staphylococcus aureus, an agent which forms stable ≈1.5 nm channels that limit exchange to molecules of approximately 1000 Da (4). Such pores will admit nucleotides and labeled residualizing probes without loss of cell proteins, a desirable, possibly necessary, condition for evaluating autophagically-mediated proteolysis.

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP)

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizzuto et al. (1995) Curr. Biol. 5, 635–642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.

Insight the mechanism of ferroptosis inhibition by ferrostatin-1

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and hence prevented by GPx4. Ferrostatin-1 (Fer-1) inhibits ferroptosis more efficiently than phenolic antioxidants. Previous studies on the reaction of Fer-1 adopted the kinetic test where a diazo-compound generates the hydroperoxyl radical to be reduced. However, this “chain breaking” effect is not satisfying for ferrous iron dependent peroxidation. New chain reactions, indeed, are primed from hydroperoxides produced by the antioxidant. On liposomes containing traces of lipid hydroperoxides and exposed to ferrous iron we disclosed by oxi-lipidomics the species produced and deduced the pattern of radical reactions. Although Fer-1 inhibits peroxidation, the pattern of oxidized species produced from pre-existing hydroperoxides was practically identical to that observed following exhaustive lipid peroxidation. This supported the notion that the anti-ferroptotic activity of Fer-1 descends from the scavenging of alkoxyl radicals generated by ferrous iron from lipid hydroperoxides. Thereof, Fer-1, in the presence if iron, eliminates lipid hydroperoxides and this produces the same anti-ferroptotic effect as GPx4, although generating a series of oxidized species but not just hydroxy fatty acids derivatives.