Ana Virel

Modulated Growth of Nanoparticles. Application for Sensing Nerve Gases

Hydrolysis of acetylthiocholine mediated by acetylcholine esterase yields the thiol-bearing compound thiocholine. At trace concentrations, thiocholine modulates the growth of Au-Ag nanoparticles on seeding gold nanoparticles in the presence of ascorbic acid. Inhibition of the enzyme by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) or by diethyl p-nitrophenyl phosphate (paraoxon) produces lower yields of thiocholine, promoting the catalytic growth of Au-Ag nanoparticles. Here, we describe the development of a simple and sensitive colorimetric assay for the detection of AChE inhibitors.

Analytical Applications of Enzymatic Growth of Quantum Dots

We have developed an analytical assay to detect the enzymatic activity of acetylcholine esterase and alkaline phosphatase based on the generation of quantum dots by enzymatic products. Acetylcholine esterase converts acetylthiocholine into thiocholine. The latter enhances the rate of decomposition of sodium thiosulfate into H(2)S, which in the presence of cadmium sulfate yields CdS quantum dots showing a time dependent exponential growth, typical of autocatalytic processes. This assay was also applied to detect acetylcholine esterase inhibitors. Alkaline phosphatase hydrolyzes thiophosphate and yields H(2)S, which instantly reacts with Cd(2+) to give CdS quantum dots. The formation of CdS quantum dots in both reactions was followed by fluorescence spectroscopy and showed dependence on the concentration of enzyme and substrate.

Magnetic Resonance Imaging (MRI) to Study Striatal Iron Accumulation in a Rat Model of Parkinson's Disease

Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson’s disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.

An altered blood–brain barrier contributes to brain iron accumulation and neuroinflammation in the 6-OHDA rat model of Parkinson’s disease

Brain iron accumulation is a common feature shared by several neurodegenerative disorders including Parkinsons disease. However, what produces this accumulation of iron is still unknown. In this study, the 6-hydroxydopamine (6-OHDA) hemi-parkinsonian rat model was used to investigate abnormal iron accumulation in substantia nigra. We investigated three possible causes of iron accumulation; a compromised blood-brain barrier (BBB), abnormal expression of ferritin, and neuroinflammation. We identified alterations in the BBB subsequent to the injection of 6-OHDA using gadolinium-enhanced magnetic resonance imaging (MRI). Moreover, detection of extravasated IgG suggested that peripheral components are able to enter the brain through a leaky BBB. Presence of iron following dopamine cell degeneration was studied by MRI, which revealed hypointense signals in the substantia nigra. The presence of iron deposits was further validated in histological evaluations. Furthermore, iron inclusions were closely associated with active microglia and with increased levels of L-ferritin indicating a putative role for microglia and L-ferritin in brain iron accumulation and dopamine neurodegeneration.

Quantification of prothrombin in human plasma amplified by autocatalytic reaction

By site directed mutagenesis, we have produced recombinant mutants of human and mouse prethrombin-2 which are able to convert themselves autocatalytically into α-thrombin. We also have created a new method to amplify the signal of bioanalytical assays based on the autocatalytic activation of these mutated proenzymes. The activation of the mutants by active α-thrombin triggers an autocatalytic reaction which leads to more active thrombin resulting in the amplification of the readout signal. Addition of mutated mouse prethrombin-2 into the conventional assay for prothrombin level in human plasma, employing ecarin and the fluorogenic substrate, resulted in improvement of the detection limit by 2 orders of magnitude.

Magnetic resonance imaging as a tool to image neuroinflammation in a rat model of Parkinson's disease--phagocyte influx to the brain is promoted by bilberry-enriched diet.

Neuroinflammation is a chronic event in neurodegenerative disorders. In the rat model of Parkinsons disease, including a striatal injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA), antioxidant treatment affects the inflammatory process. Despite a heavy accumulation of microglia early after the injury, dopamine nerve fibre regeneration occurs. It remains unclear why this heavy accumulation of microglia is found early after the lesion in antioxidant‐treated animals, or even more, what is the origin of these microglia. In this study magnetic resonance imaging (MRI) was used to elucidate whether the inflammatory response was generated from the blood or from activated brain microglia. Superparamagnetic iron oxide (SPIO) nanoparticles were injected intravenously prior to a striatal 6‐OHDA injection to tag phagocytes in the blood. Rats were fed either with bilberry‐enriched or control diet. T2*‐weighted MRI scans were performed 1 week after the lesion, and hypointense areas were calculated from T2*‐weighted images, to monitor the presence of SPIO particles. The results revealed that feeding the animals with bilberries significantly promoted accumulation of blood‐derived immune cells. Gadolinium‐enhanced MRI demonstrated no difference in leakage of the blood–brain barrier independent of diets. To conclude, bilberry‐enriched diet promotes an influx of periphery‐derived immune cells to the brain early after injury.