Studio del lisozima nel contesto della nefropatia diabetica

Study of lysozyme in the context of diabetic nephropathy Introduction Lysozyme (LZ), enzyme known for its antimicrobial and immunomodulant activity, has recently shown to be effective in the context of diabetic nephropathy, one of the major complications associated to diabetes and one of the major cause of end stage kidney disease in Western countries. Experimental evidences strictly link the development and progression of the diabetic nephropathy to the enormous production and accumulation of Advanced Glycation Endproducts (AGEs). AGEs are a chemically heterogeneous group of macromolecules which formation is dramatically increased in diabetic patient mainly due to chronic hyperglicaemia. Nevertheless, several factor, such oxidative and carbonyl stress and reduced renal clereance seem to be related to the increase of the production and accumulation of AGEs. The first step of AGEs formation starts in presence of reducing sugars, as glucose, which are able to interact with free amino group (Maillard reaction), forming Schiff base. The rearrangements from the Schiff base leads to the formation of Amadori products, that are eventually oxidazed, leading to the formation of AGEs. AGEs binding to structural proteins and deposition in tissues alter functions, leading to stiffening, enhanced local cytokine production and is a marker of cumulative metabolic stress. At cellular level, AGEs-induced events, depend mainly by their interaction with specific receptor, among which RAGE, is the most well characterized. AGE-RAGE interactions determine a number of events, such as an increased production of reactive oxygen species (ROS), induction of pro-inflammatory mediators, upregulation of RAGE receptor. In the last decades, it has been shown that hen egg white LZ is able to act effectively as AGEs scavenger (Kd= 50 nM). In in vivo models it was demonstrated that orally administered microencapsulated LZ, can act prevent the insurgence of a number of relevant early manifestations of diabetic nephropathy, such as microalbuminuria and glomerular hypertrophy. Aim The aim of the present work is to develop an in vitro model for studying the molecular mechanisms responsible for the nephroprotective activity of LZ, through the identification and validation of an adequate cell line and a marker. In order contribute to understand the higher in vivo effectiveness of microencapsulated LZ compared to an equidose of “free” LZ, a sophisticated model of incannulated rat will be developed. Results and discussion An adequate cell model was selected among three different cell lines: a primary culture of endothelial cells (ADMEC), due to the implication of vascular tissue in diabetes and two proximal tubular cell lines, LLC-PK1 (porcine cells) and HK-2 (human cells) due to the tubular involvement in the context of diabetic nephropathy. HK-2 cell line was selected on the basis of its dose-dependent sensibility to the AGE treatments, quantified, in terms of viability, by means of MTT test. One of the most relevant AGE-induced effect is the induction of inflammatory response. A pivotal event in the context of inflammation is represented by macrophage recruitment. For this reason, it was performed a migration assay, using monocyte U937 differentiated in macrophage, toward a stimulus represented by supernatants obtained treating HK-2 cells with AGE e LZ. AGE treatment induced a significant increased (+50%) of the migrated macrophage. The co-treatment significantly prevented the migration induction. LZ treatment, as expected, did not modify migratory ratio. It is known that cytokine play a crucial role in macrophage recruitment. Interleukin-6 (IL-6), for what diabetic nephropathy is concerned, has a pivotal role. On the basis of this evidence, the IL-6 mRNA levels after AGE and LZ treatments were measured. According with expectations, AGE induced a significant IL-6 mRNA increase after 24 h of treatment. LZ exposition did not induced any modifications, whereas the co-treatment with AGE and LZ showed that LZ prevented IL-6 mRNA increase. In order to confirm the effectiveness of LZ toward AGE-induced IL-6 increase, an ELISA assay was performed. LZ was able to significantly and dose-dependently reduce the AGE-induced release of IL-6 in the supernatants. Several mechanisms have been reported to be capable to induce IL-6 release. The mechanisms that determine IL-6 release are several. On the basis of the recognized crucial role attributed to RAGE receptor, it was tested the capacity of LZ to influence RAGE mRNA levels, by means of RT-PCR technique. The results showed no variations of RAGE levels after exposition to AGE and LZ for 24 up to 96 h. The induction of ROS represents one of the events attributable to AGEs. Nevertheless, on the cellular model employed, this parameter did not show any significant increase. Considering that LZ activity is associated also to its AGE scavenging action, it was evaluated by means of confocal microscopy, if the presence of LZ could influence the AGE capacity to enter inside the cell. The images obtained by means of this technique showed that AGEs and LZ are both able to enter in the cells. In order to investigate the co-treatment effect a cytometry analysis was performed. The results suggest that LZ is not able, in this cellular model, to prevent the entrance on AGEs inside the cells, excluding that LZ scavenging action could be the cause of preventive action of in vitro IL-6 production. Subsequent studies were performed on pathways potentially involved in the action of LZ toward AGEs, such as MAPK cascade (p38) and lysosomial degradation. Preliminary data showed a possible involvement of p38. In fact, AGEs showed the capacity to increase phosphorylation level of this protein, effect reduced by the co-treatment with an equimolar dose of LZ. Promising are also the first results obtained from the lysosomial localization study of AGE, in presence of LZ. In fact, the qualitative analysis of the obtained figures confirms the hypothesis that LZ is able to bring AGEs in lysosomes, supporting their degradation. Taken together these data do not exclude stochastically that LZ action is mainly antidotic. However, the LZ capacity to act toward pivotal mediators of AGE-induced effects, such as IL-6 and p38, open new work perspectives for what LZ action is concerned, allowing to speculate its use as cooperating drug in the control of inflammation in the context of diabetic nephropathy. In order to investigate if the higher effectiveness of orally administered microencapsulated LZ was due to the capacity of microparticles to deliver more efficiently the LZ in the blood stream an in vivo test was performed. The results showed that microparticles can contribute significantly to deliver LZ more efficiently to seru

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Energy efficiency in smart buildings requires distributed sensing infrastructure to monitor the power consumption of appliances, machines and lighting sources. The analysis of current and voltage waveforms is fundamental for gathering diagnostic information about the power quality and for reducing power wastage. Moreover, it enables Non-intrusive Load Monitoring (NILM), which is the process of disaggregating a household's total electricity consumption into its contributing appliances, by analysing the voltage and current changes. In this paper, an innovative full Energy-neutral (i.e. battery free) and Non-intrusive Wireless Energy Meter (NIWEM) is presented to measure current, voltage and power factor. As key features, the NIWEM is completely non-invasive and it can self-sustain its operations by harvesting energy from the monitored load. It also features a standard (Zigbee) wireless interface for communication with the smart-building system. Experimental results have confirmed that complete energy sustainability can be achieved also with very low-power loads

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‘Coming back Home’ . Il Modello Virtuale della Statua Romana di Asclepio del Museo di Siracusa (Italia)

The colossal torso of the god Asclepius, kept into the Castello Maniace during the Spanish domination of Sicily, is now one of the most significant examples of roman statuary in the Syracuse Museum. The recent restoration of the Castello Maniace has been celebrated in 2008 with an exhibition of archaeological finds obtained in the various excavations of the castle. The statue of Asclepius, which had for centuries been a part of the architecture there, should have been the most significant piece on display. However, its large size and frailty made such a temporary move inadvisable, and it was decided that a plaster cast should be made and exhibited instead. A team of archaeologists and information technicians from the Archeomatica Project of Catania University were able to contribute to this task by creating a 3D model of the statue through the use of laser scanning techniques