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ABSTRACT Introduction: Transcranial Doppler ultrasonography (TCD) is a technique that allows measurement of blood flow from the basal intracerebral vessels. It is relatively inexpensive, non-invasive, can be performed at the bedside, and allows monitoring in acute emergency settings and for prolonged periods with a high temporal resolution, making it ideal for studying the haemodynamics within the intracranial arteries in neuro-Behcet's disease (NBD) and neuro-psychiatric lupus (NPSLE). Our aim was to assess the cerebral haemodynamic patterns in patients with NBD and NPSLE using TCD, while brain lesions were examined using magnetic resonance imaging (MRI). Material and methods: Case-control prospective study of 30 neuro-Behcet's disease patients, 25 neuro-psychiatric lupus patients and 26 healthy age-matched volunteers. All patients and healthy controls were examined by TCD. Only the groups of patients underwent cranial magnetic resonance imaging (MRI). Results: Transcranial Doppler (TCD) values for middle cerebral artery (MCA), anterior cerebral artery (ACA), posterior cerebral artery (PCA), vertebral artery (VA) and basilar artery (BA) in NBD, NPSLE and control groups were measured. The results showed that there was a significant decrease in mean blood flow velocities in all the arteries examined in NBD and NPSLE patients. There was also a significant increase in the pulsatile index of PCA, VA and BA between NBD and NPSLE patients. The same results were obtained when comparing NBD versus controls. However, there was no significant difference between the NPSLE patients and the control group. The MRI lesions described were parenchymal lesions in 14 patients (46.7%), and vascular lesions in 4 patients (13.3%). Vascular lesions co-existed with parenchymal lesions (mixed lesion). Parenchymal lesions were in white matter (40%), thalamus (26.7%), brain stem (26.7%) and cerebellum (20%). While, in NPSLE, 23 patients were normal (92%) and only two patients had a vascular lesion (8%). Conclusion: There was a significant decrease in mean blood flow and a significant increase in the pulsatile index among both NBD and NPSLE patients, according to the TCD values.

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Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ2. From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.

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We report the development of a new analytical method for the quantification of N-(phosphonomethyl)glycine (glyphosate) and (aminomethyl)phosphonic acid (AMPA) by combining spectrofluorimetry and multivariate calibration. In this study, fluorescence spectroscopy was used to quantify glyphosate and AMPA, which were previously derivatized with the fluorogenic reagent: 4-chloro-7-nitrobenzofurazan (NBD-Cl). Fluorescence excitation-emission matrices (EEM) were recorded by exciting between 400 and 500 nm, and measuring the emission between 500 and 610 nm. The second-order data obtained were processed using the Multivariate Curve Resolution with Alternating Least Square (MCR-ALS) methodology. The developed method was used to predict different concentrations of glyphosate and AMPA in validation samples. In addition, the presence of the herbicide was evaluated in real samples: a commercial formulation and a water sample from a cultivated area. For this purpose, the standard addition method was used to study the matrix effect in each case. The ranges of working concentrations obtained for this new method are in agreement with the amounts found in surface water samples near a direct sowing soybean growing region in Argentina.

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The toxic effects of miltefosine on the epithelial cells of the gastrointestinal tract and its hemolytic action on erythrocytes have limited its use as an antileishmanial agent. As part of our search for new strategies to overcome the side effects of miltefosine during the treatment of leishmaniasis, we have developed stable miltefosine-loaded lipid nanoparticles in an attempt to reduce the toxic effects of the drug. We have evaluated lipid nanoparticles containing varying amounts of miltefosine and cholesterol, prepared by sonication, in terms of their physicochemical properties, preliminary stability, hemolytic potential toward erythrocytes, and cytotoxicity to macrophages and to promastigote and amastigote forms of Leishmania (L.) chagasi. Miltefosine loading into lipid nanoparticles was 100% for low drug concentrations (7.0 to 20.0mg/mL). Particle size decreased from 143nm (control) to between 43 and 69nm. From fluorescence studies, it was observed that the presence of miltefosine and cholesterol (below 103µM) promoted ordering effects in the phospholipid region of the nanoparticles. The formulation containing 15mg/mL miltefosine was stable for at least six months at 4°C and in simulated gastrointestinal fluids, and did not promote epithelial gastrointestinal irritability in Balb/C mice. When loaded into lipid nanoparticles, the hemolytic potential of miltefosine and its cytotoxicity to macrophages diminished, while its antiparasitic activity remained unaltered. The results suggested that miltefosine-loaded lipid nanoparticles may be promising for the treatment of leishmaniasis and might be suitable for oral and parenteral use.

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Tamoxifen, an antineoplastic agent, is active in vitro and in vivo against the parasitic protozoa Leishmania. As part of our efforts to unravel this drug's mechanisms of action against the parasite and understand how resistance could arise, we tried to select tamoxifen-resistant Leishmania amazonensis. Three different strategies to generate tamoxifen resistant mutants were used: stepwise increase in drug concentration applied to promastigote cultures, chemical mutagenesis followed by drug selection and treatment of infected mice followed by selection of amastigotes. For amastigote selection, we employed a method with direct plating of parasites recovered from lesions into semi-solid media. Tamoxifen resistant parasites were not rescued by any of these methods. Miltefosine was used as a control in selection experiments and both stepwise selection and chemical mutagenesis allowed successful isolation of miltefosine resistant mutants. These findings are consistent with a multi-target mode of action to explain tamoxifen's leishmanicidal properties. Considering that drug resistance is a major concern in anti-parasitic chemotherapy, these findings support the proposition of using tamoxifen as a partner in drug combination schemes for the treatment of leishmaniasis.

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