Application of DIGE and mass spectrometry in the study of type 2 Diabetes Mellitus (T2DM) mouse models

Knowledge of the differences between the amounts and types of protein that are expressed in diseased compared to healthy subjects may give an understanding of the biological pathways that cause disease. This is the reasoning behind the presented protocol, which uses difference gel electrophoresis to discover up‐ or down‐regulated proteins between mice of different genotypes, or of those fed on different diets, that may thus be prone to develop diabetes‐like phenotypes. Subsequent analysis of these proteins by tandem mass spectrometry typically facilitates their identification with a high degree of confidence

GIS and remote sensing for post-dictive analysis of archaeological features. A case study from the Etnean region (Sicily)

This article illustrates the potential of multispectral satellite data for archaeological scope in the volcanic area of Mount Etna (Sicily, Italy). In particular, by adopting a post-dictive approach, GIS and FOSS technology was used to analyse different indices derived from World-View-2 multispectral data. The selected examples (two circular buildings and a wall-structure) illustrate successes and challenges of our method. The results indicate that NIR-1 and RED-EDGE are undoubtedly the most useful, while NDVI and SRI are the best performing indices

Dosimetria tramite Risonanza Elettronica di Spin (ESR) in RadioTerapia IntraOperatoria (IORT): misure di Output Factors e simulazioni Monte Carlo-GEANT4

La RadioTerapia IntraOperatoria (IORT) è una modalità di trattamento in cui una singola dose di radiazioni è impartita direttamente al letto tumorale o al tumore durante l'intervento chirurgico, evitando di colpire i tessuti sani circostanti. La fabbricazione di acceleratori lineari mobili per elettroni dedicati alla IORT ha permesso una grande diffusione di questa tecnica radioterapica. Lo scopo di questo lavoro è il confronto tra la risposta di dosimetri di alanina letti tramite Risonanza Elettronica di Spin (ESR) e di camere a ionizzazione Markus per le misurazioni degli Output Factors (OFs) di fasci di elettroni prodotti da un acceleratore lineare utilizzato per la IORT. Gli OFs dei fasci di elettroni convenzionali ad alta energia sono normalmente misurati utilizzando camera di ionizzazione secondo protocolli dosimetrici internazionali. Tuttavia, i fasci elettronici utilizzati in IORT presentano caratteristiche quali (quali impulso di dose, spettro energetico e distribuzione angolare molto diversa dai fasci solitamente utilizzate in radioterapia esterna), per cui l'applicazione diretta di protocolli dosimetrici internazionali può introdurre ulteriori incertezze dosimetriche. Gli OFs ottenuti mediante dosimetri di alanina letti tramite ESR sono stati confrontati con quelli ottenuti con camere a ionizzazione di tipo Markus. Il confronto è stato completato da simulazioni Monte Carlo utilizzando l’applicazione dedicata alla IORT di Geant4 che consente di ottenere informazioni dettagliate sulla distribuzione di dose

Biological surface properties in extracellular vesicles and their effect on cargo proteins

Ultracentrifugationon sucrose density gradientappears to be the best purification protocol for extracellular vesicle (EVs) purification. After this step, to reduce disulfide bridges linking exogenous proteins to the vesicles, the collected samples are routinely washed and treated with dithiothreitol (DTT). Such incubations are performed at temperatures ranging from room temperature up to 95\u2009\ub0C, with either Tris or PBS as buffers. We re-investigated these steps on both exosomes and microvesicles purified from blood (serum) and urine by electrophoretic separation, silver staining and western blots analysis. Data confirm that an extra centrifugation on a sucrose cushion can effectively eliminate contaminants. Tris buffer (50 Mm) and \u3b2-mercaptoethanol as a reducing agent at room temperature dramatically improved either sample cleaning. By contrast, especially for exosomes PBS buffer and DTT, above 37\u2009\ub0C, caused massive protein aggregations, yielding blurred SDS-PAGE gels in both samples. Immuno-blot analyses demonstrated that in PBS-DTT contamination with albumin (in serum) or with uromodulin (in urine) occurs. DTT, likely due to its two-SH groups, might form scrambled SS-bonds promoting EVs interaction with environmental macromolecules via disulphide bridges. Therefore, to obtain maximum vesicle purity for biomarker investigations and to maximize both presence of EVs proteins and their accessibility, use of DTT is not recommended

Oxidative stress as a primary risk factor for brain damage in preterm newborns

The risk of oxidative stress is high in preterm newborns. Room air exposure of an organism primed to develop in a hypoxic environment, lacking antioxidant defenses, and subjected to hyperoxia, hypoxia, and ischemia challenges the newborn with oxidative stress production. Free radicals can be generated by a multitude of other mechanisms, such as glutamate excitotoxicity, excess free iron, inflammation, and immune reactions. Free radical-induced damage caused by oxidative stress appears to be the major candidate for the pathogenesis of most of the complications of prematurity, brain being especially at risk, with short to long-term consequences. We review the role of free radical oxidative damage to the newborn brain and propose a mechanism of oxidative injury, taking into consideration the particular maturation-dependent vulnerability of the oligodendrocyte precursors. Prompted by our observation of an increase in plasma Adenosine concentrations significantly associated with brain white matter lesions in some premature infants, we discuss a possible bioenergetics hypothesis, correlated to the oxidative challenge of the premature infant. We aim at explaining both the oxidative stress generation and the mechanism promoting the myelination disturbances. Being white matter abnormalities among the most common lesions of prematurity, the use of Adenosine as a biomarker of brain damage appears promising in order to design neuroprotective strategies

Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

Alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs) such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations). In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far

Proteome profile of peritoneal effluents in children on glucose- or icodextrin-based peritoneal dialysis

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Active focal segmental glomerulosclerosis is associated with massive oxidation of plasma albumin

The basic mechanism for idiopathic FSGS still is obscure. Indirect evidence in humans and generation of FSGS by oxidants in experimental models suggest a role of free radicals. In vitro studies demonstrate a main role of plasma albumin as antioxidant, its modification representing a chemical marker of oxidative stress. With the use of complementary liquid chromatography electron spray ionization tandem mass spectrometry (LC-ESI-MS/MS) and biochemical methods, plasma albumin was characterized in 34 patients with FSGS; 18 had received a renal transplant, and 17 had IgM mesangial deposition. Patients with FSGS that was in remission or without recurrence after transplantation had normal plasma albumin, and the same occurred in patients with primary and secondary nephrites and with chronic renal failure. In contrast, patients with active FSGS or with posttransplantation recurrence had oxidized plasma albumin. This finding was based on the characterization of albumin Cys 34 with an mass-to-charge ratio of 511.71 in triple charge that was consistent with the formation of a cysteic acid carrying a sulfonic group (alb-SO3-). The exact mass of albumin was increased accordingly (+48 Da) for incorporation of three oxygen radicals. Direct titration of the free sulfhydryl group 34 of plasma albumin and electrophoretic titration curves confirmed loss of free sulfhydryl group and formation of a fast-moving isoform in all cases with disease activity. This is the first demonstration of in vivo plasma albumin oxidation that was obtained with an adequate structural approach. Albumin oxidation seems to be specific for FSGS, suggesting some pathogenetic implications. Free radical involvement in FSGS may lead to specific therapeutic interventions