Detection methodologies for microRNA biomarker profiling

Owing to their pivotal role as expression regulators, microRNAs (miRNAs) have different physiological roles and can be involved in the onset and progression of several diseases. For this reason, their altered presence can be predictive of a pathological state. Furthermore, the ubiquitous presence of these short RNA sequences in basically all body tissues and fluids makes them elite candidates as biomarkers. With this concept in mind, it is fundamental to have effective, sensitive, and selective techniques to perform their accurate detection and profiling. The goal of this chapter is to summarize the pros and cons of some of the available detection approaches used for miRNA profiling, starting from the most common unamplified/amplified probe-based methods to the advanced techniques based on biosensors and bioassays

Etching of InP-based MQW laser structure in a MOCVD reactor by chlorinated compounds

Four different chlorinated compounds: 2-chloropropane, dichloromethane, chloroform and carbon tetrachloride have been used to etch InGaAsP/InP MQW laser structures partially masked. Etching experiments were performed in a home-made LP-MOCVD reactor with argon or argon+hydrogen as carrier gas, using phosphine (PH3) or tertiarybutylphosphine (TBP) to prevent thermal decomposition. The etching temperature as well as the chlorinated compound flow were varied to obtain the best trade-off between etch rate and surface morphology. The optimized experimental conditions were applied to etch mesa stripes in a SCH-MQW laser structure, for the first time to our knowledge, followed by lateral InP : Fe regrowth in the same step. Threshold current as low as 4 mA (best value)-6 mA (typical value) and differential quantum efficiency higher than 20% for SI-BH MQW laser have been achieved

Effect of normothermic ("Warm") hypoxia on oxidative stress and metabolism in isolated/perfused rat liver: histochemical and biochemical evaluations.

It is generally believed that the damage induced by “warm” ischemia is negligible with respect to that caused by reperfusion and that Reactive Oxygen Species (ROS) are the main effectors of tissue damage (Le Masters & Thurman, 1993). In order to check the presumed poor response and ROS production of the various liver cell populations to lack of oxygen and nutrients, we used histochemical and biochemical techniques to document the response of isolated rat liver perfused with N2- or O2-saturated medium at 37°C for up to 2 hours. We analyzed the perfusate for LDH activity released from dead cells and for thiobarbituric acid reactive species (TBARS) derived from oxidative stress and measured bile flow as index of liver metabolism. Assays were made every 10 min. Two significant time points of tissue damage (40 and 70 min) were selected to perform the histochemical assays. These comprised the demonstration of: (a) ROS production, with Kerver et al. (1997) Mn2+-Co3+-DAB technique; (b) the putative diaphorase activity of Nitric Oxide Synthase (NOS), with a technique improved in our laboratory (Freitas et al., 2002); and (c) the activity of several enzyme activities linked to liver metabolism, namely Lactate Dehydrogenase (LDH), Succinate Dehydrogenase (SDH), Alkaline Phosphatase (AlkPh), Purine Nucleoside Phosphorylase (PNP) and Xanthine Oxidoreductase (XOR; comprising both the reductase and the oxidase forms of the enzyme), with standardized techniques (Van Noorden & Frederiks). Trypan Blue uptake was used as indicator of cell death. A scoring method was used to compare semi-quantitatively the response of hepatocytes, sinusoidal and biliary cells. Normoxic perfusion had negligible effects. By contrast, warm hypoxia caused lack of bile flow, hepatocyte staining by Trypan Blue in the midzone, alteration of the activity of all enzymes, increased ROS production by hepatocytes and sinusoidal cells (in keeping with high TBARS concentration in the perfusate) and release of LDH to the perfusate (in keeping with LDH loss from mid-zonal hepatocytes). ROS appeared to be produced mostly as by-products of the mitochondrial electron transfer chain in hepatocytes and during (auto)phagocytosis in sinusoidal cells and pericentral hepatocytes. NOS activity in Ito cells appeared to be correlated to sinusoid dilatation and PNP activity to be a sensitive marker of damage to sinusoidal and bile duct cells. In conclusion, our results suggest that the vulnerability of the liver to warm hypoxia is lobular zone and cell type-dependent and that damage to the parenchyma is more serious than that expected from biochemical data alone. (Funds from FAR-UniPV; MIUR-COFIN 1999, 2001

Monitoring harvesting degree of red Scarlet apples using DR-UV-Vis and NIR spectroscopy

Establishing the optimal harvest time is a fundamental issue for apple fruit industry, since fruit shelf life and quality are related to the ripening stage at harvest. Ripening indexes determination through destructive analysis is invasive for fruits and is limited to a low sample number. This work is focused on the study of red apples ripening (Scarlet variety), with the aim of better identifying harvest date characterizing each stage of fruit ripening on trees using classical analysis and non-destructive analysis. UV-Vis spectroscopy is performed in diffuse reflectance mode to monitor variation of chlorophyll concentration in the skin of apples during ripening on trees; in fact, in the case of fruits as red apples the colour variation is hardly visible as ripening goes on, and ground colour cannot be considered a valid tool of ripening identification. Conversely chlorophyll monitoring using DRS-UVVis spectroscopy appears to be a reliable indicator of the ripeness leve

Ischemia/reperfusion effects on pig liver metabolism and autofluorescence.

Ischemia/reperfusion effects on pig liver metabolism and autofluorescence

MICROENVIRONMENT INFLUENCE ON STEM CELLS: PRE-CARCINOGENESIS FEATURES IN THE LIVER OF TRANSGENIC MMTV-NEU (erbB-2) MICE BEARING MAMMARY TUMOR.

Attention is called to a non-negligible risk in the Regenerative Medicine setting using bone marrow-derived stem cells of inducing a tumor in the organ to be repaired or else in a different organ. Premises for this concern came from a recent research of ours on a transgenic animal model of human mammary carcinoma (MMTV-neu (erbB-2 transgenic mice). In these animals several lobular-zone dependent features of initial liver carcinogenesis were detected. In portal and sub-capsular regions CD34+ cells were seen followed, in time, by extramedullary hemopoiesis. In portal tracts liver oval cells were identified followed by ductular reaction, while portal hepatocytes expressed CK19, AFP and GGT (markers of hepatocytes in the embryo). In the mid-zone and centrolobular regions Large cell Dysplasia was seen. The fetal portal microenvironment might be better suited than that of the adult to the activation of liver cell progenitors. By contrast, dysplasia suggests polyploidy and terminal differentiation. Taken together, these observations might be induced by sustained metabolic stress induced by the tumor on the liver. It is speculated that hemopoietic precursors, recruited by the tumor for angiogenic purposes, find in the liver a permissive environment for their arrest and differentiation. Attention is called for the fact that the final fate of stem/progenitor cells depends on their microenvironment and that currently Cancer is considered as a stem-cell pathology. The conclusion is drawn that the final consequence of the activation of such an ancestral and non-specific system of repair might be diversified according to the final destination of the activated precursors