Leaf apoplastic proteome composition in UV-B treated Arabidopsis thaliana mutants impaired in extracellular glutathione degradation

In plants, environmental perturbations often result in oxidative reactions in the apoplastic space, which are counteracted for by enzymatic and non-enzymatic antioxidative systems, including ascorbate and glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform GGT1 bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with UV-B radiation and studied in redox altered ggt1 mutants. Elevated UV-B has detrimental effects on plant metabolism, plasma membranes representing a major target for ROS generated by this harmful radiation. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in apoplastic protein composition. We then compared the expression changes resulting from the mutation and from the UV-B treatment. Rearrangements occurring in apoplastic protein composition suggest the involvement of hydrogen peroxide, which may ultimately act as a signal. Other important changes related to hormonal effects, cell wall remodeling, and redox activities are also reported. We argue that oxidative stress conditions imposed by UV-B and by disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data shown here are associated with the article from Trentin et al. [1]; protein data have been deposited to the PRIDE database [2] with identifier PXD001807

CLASSIFICAZIONE DI MELANOMI BASATA SU ELABORAZIONE DI IMMAGINI E UTILIZZO DI RETI NEURALI

Durante le ultime due decadi, si è osservato un aumento della frequenza di un particolare tumore della pelle, il melanoma. A causa della mancanza di adeguate terapie per una classe di questi tumori particolarmente pericolosa, quelli metastatici, il miglior trattamento attuale risulta essere ancora la diagnosi precoce e la tempestiva asportazione chirurgica del tumore primario. Una nuova tendenza, assecondata da molti dermatologi, è quella di coinvolgere nella lotta al melanoma anche i medici generici, che rappresentano la prima linea di cura tra i pazienti e i centri specialistici. Ad essi infatti dovrebbe essere affidato, come avviene in altre discipline della prevenzione medica, lo screening primario delle lesioni pigmentate della pelle in modo da individuare immediatamente tra i loro pazienti quelli che hanno delle lesioni sospette e necessitano di una visita specialistica dermatologica, e quelli che hanno solamente delle lesioni banali e non corrono alcun tipo di rischio. Oggi, grazie alle nuove tecnologie informatiche e grazie ai miglioramenti dei dispositivi per l’acquisizione delle immagini digitali, abbiamo un nuovo e promettente approccio nella diagnosi delle lesioni pigmentate: l’analisi automatica delle immagini dermoscopiche, a supporto del personale medico non specialistico per la diagnosi precoce dei casi sospetti di melanoma. In questa tesi è stato quindi affrontato il problema di realizzare un sistema informatico capace di classificare in maniera automatica le lesioni cutanee in benigne e sospette, partendo dalle loro immagini dermoscopiche. L’approccio seguito si basa sulla misura di opportune caratteristiche morfologiche delle lesioni cutanee pigmentate, come il colore, la simmetria e la forma, estratte mediante tecniche di elaborazione delle immagini, per ottenere la classificazione mediante reti neurali multistrato

NBS1 Heterozygosity and Cancer Risk

Biallelic mutations in the NBS1 gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBS1 gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBS1 heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBS1 gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBS1 protein, which are present in a large superfamily of proteins, and are recognized as major mediators of processes related to cell-cycle checkpoint and DNA repair

LA MODULAZIONE DEI LIVELLI DI GLUTATIONE COME STRATEGIA DI ATTACCO NELLE INTERAZIONI OSPITE-PARASSITA

Insect studies, dealing with parasitism of aphids, have shown that the disruption of host glutathione (GSH) pool and metabolisms significantly contributes to its physiological regulation and castration. The parasitic wasp Aphidius ervi injects into host aphids a venom containing large amounts of a gamma-glutamyltransferase (Ae-GGT) enzyme, which causes a depletion of GSH primarily involving ovarian tissue. Injected Ae-GGT in fact consumes substrate GSH, which ultimately triggers apoptosis. Studies on virulence factors of microrganisms have documented that the invasion strategies of selected pathogenic bacteria also target host GSH metabolism. Indeed, it has been shown that GGT activity of Helicobacter pylori and H. suis, the agents responsible of peptic ulcer, can exert antiproliferative and pro-apoptotic effects in gastric epithelial cells. By confocal microscopy, H. suis outer membrane vesicles (OMV) 12 submicroscopic structures 20-50 nm in diameter, budding from the cell surface 12 were identified as carriers of H. suis GGT, capable of delivering the enzyme to the deeper mucosal layers. In association with such membranous structures, active GGT from H. suis in fact translocates across the epithelial layers and can access lymphocytes residing in the gastric mucosa, resulting in the inhibition of lymphocyte proliferation, i.e., a perturbation of host immunity and a facilitation of bacterial infection. Cellular GSH appears, thus, to represent a conserved target for parasitic (micro)organisms which aim at altering host redox homeostasis to weaken its immune defenses, using GGT as a key-element of a virulence strategy. Taking into account the \u201cparasitic\u201d behavior exhibited by malignant cells spreading across tissues and organs of the patient (the \u201chost\u201d). GGT activity is in fact expressed in a number of malignant tumors, and expression levels often increase along with progression to more invasive phenotypes. Now, active GGT can be released from cells, including cancer cells, in association with submicroscopic vesicles resembling exosomes. The similarity of such structures with GGT-rich OMV particles of H. pylori and H. suis is indeed obvious. GGT activity of cancer cells can affect intracellular redox equilibrium, and produces in addition significant extracellular effects, e.g. on the redox status and ligand binding affinity of cell surface receptors related with cell survival/apoptosis balance. Thus, GGT-rich exosomes shed by cancer cells can produce in host\u2019s surrounding tissues effects comparable to those reported for Ae-GGT or Helicobacter GGT, possibly resulting in facilitation of malignant cells survival and diffusion

Energy and Seismic Rehabilitation of RC Buildings through an Integrated Approach: An Application Case Study

The high number of existing buildings in Italy without adequate seismic and thermal performances requires the definition of integrated retrofitting techniques in order to improve the seismic performance and to reduce energy losses at the same time. On one hand, an integrated approach appears mandatory considering that improving only the energy efficiency of nonseismic buildings leads to an increase of their exposure and, therefore, of their risk in the case of seismic events. On the other hand, seismic strengthening without an adequate thermal assessment and rehabilitation could compromise living comfort and energy maintenance costs. In this context, an application of integrated approach for the rehabilitation of reinforced concrete (RC) existing buildings has been proposed referring to a case study representative of the Italian building stock. Different configurations of infill panels have been considered in order to analyze both energy and seismic performance. Monthly quasi-steady state and hourly dynamic models have been used for the calculation of the energy need of buildings located in different Italian climate and seismic zones. Seismic performances have been evaluated by means of incremental nonlinear dynamic analysis (IDA). As-built and post-retrofit performances have been compared in order to evaluate the effectiveness of the proposed intervention solutions

Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey

In the dairy industry, membrane filtration, is used to reduce the amount of whey waste and, simultaneously, to recover whey proteins (WP). The composition of WP can strongly affect the filtration treatment of whey, and rapid determination of WP fractions would be of interest for dairy producers to monitor WP recovery. This study aimed to develop mid-infrared spectroscopy (MIRS) prediction models for the rapid quantification of protein in sweet whey, using a validated rapid reversed phase (RP)-HPLC as a reference method. Quantified WP included alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG) A and B, bovine serum albumin, caseinomacropeptides, and proteose peptone. Validation of RP-HPLC was performed by calculating the relative standard deviation (RSD) in repeatability and reproducibility tests for WP retention time and peak areas. Samples of liquid whey (n = 187) were analyzed by RP-HPLC and scanned through MIRS to collect spectral information (900 to 4,000 cm(-1)); statistical analysis was carried out through partial least squares regression and random cross-validation procedure. Retention times in RP-HPLC method were stable (RSD between 0.03 and 0.80%), whereas the RSD of peak area (from 0.25 to 8.48%) was affected by WP relative abundance. Higher coefficients of determination in validation for MIRS model were obtained for protein fractions present in Whey in large amounts, such as beta-Lc (0.58), total identified WP (0.58), and alpha-LA (0.56). Results of this study suggest that MIRS is an easy method for rapid quantification of detail protein in sweet whey, even if better resolution was achieved with the method based on RP-HPLC. The prediction of WP in sweet whey by MIRS might be used for screening and for classifying sweet whey according to its total and individual WP contents

Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity. Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins. Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division. Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses