Dysphagia and pulmonary complications in acute cerebrovascular disease: A retrospective observational study.

INTRODUCTION: Dysphagia is a common post-stroke complication, which may result in serious pulmonary sequelae. Early detection of dysphagia and aspiration risk can reduce morbidity, mortality and length of hospitalization. OBJECTIVES: This study aims to identify association between dysphagia and acute cerebrovascular disease, and evaluate the prevalence and impact of pulmonary complications on readmissions and mortality. MATERIAL AND METHODS: Retrospective observational study based on 250 clinical records of patients with acute cerebrovascular disease: clinical history, neurological examination, imaging and Gugging Swallowing Screen in the first 48h. Patients were followed for 3 months via medical records to estimate 3-month mortality and readmissions. RESULTS: Out of 250 clinical records analyzed, 102 (40.8%) were evaluated for dysphagia. The prevalence of dysphagia was 32.4%. The risk was higher in older patients (p<0.001), in severe stroke (p<0.001) and in the hemorrhagic subtype (p=0.008). An association was found with dysarthria and aphasia (p=0.003; p=0.017). Respiratory tract infections occurred in 14.4% of all patients (GUSS group 11.8% versus no GUSS group 16.2%), and in 75% of those with severe dysphagia (p<0.001). Mortality at 3 months was 24.2% in dysphagic patients, especially high in the severe dysphagia group (75%, p<0.001). CONCLUSIONS: The type of cerebrovascular disease, NIHSS and GCS scores, age, dysarthria, and aphasia were significant associated factors to dysphagia. The prevalence of respiratory tract infections was higher in patients with no GUSS record, and no statistical significance was observed in related readmissions. Mortality at 3 months was superior in the severe dysphagia group.

Supraorbital trans-eyebrow craniotomy and fluorescence-guided resection of fronto-basal high grade gliomas.

OBJECT: To determine the effectiveness of fluorescence-guided resection of fronto-basal high grade gliomas by using the supraorbital trans-eyebrow craniotomy. METHODS: We present a single-institution experience of 6 consecutive patients presenting high grade brain glioma located on the fronto-basal area that were operated through a supraorbital trans-eyebrow craniotomy. Previous to surgery all patients were administered 20mg/kg of 5 aminolevulic acid so microscopic fluorescence-guided resection could be accomplished. Tumors were located on gyrus rectus (3 patients), medial orbital gyrus (2 patients), and anterior orbital gyrus (1 patient). RESULTS: Despite the narrow surgical corridor, fluorescence was useful in all cases. Fluorescence-guided resection allowed inclusion into the margins of resection of areas previously considered as normal under white light. Complete resection was obtained in 5 patients. No neurological postoperative new deficit was observed in this series. All six cases corresponded to glioblastoma. Only one case of superficial infection with delayed wound healing was reported as complication. All patients expressed a high level of satisfaction related to cosmetic result. CONCLUSIONS: Fluorescence-guided resection of fronto-basal high grade gliomas can be successfully achieved through supraorbital trans-eyebrow craniotomy. Benefits of supraorbital craniotomy in the management of fronto-basal high grade gliomas as well as usefulness of fluorescence-guided resection through a very narrow corridor are exposed.

Desenvolvimento e Caracterização de Nanopartículas de D, L-PLA contendo Cefoxitina / Development and Characterization of Cefoxitin Loaded D, L-PLA Nanoparticles

As nanopartículas de D, L-PLA (PLA) contendo cefoxitina (CEF) foram preparadas pelo método de emulsão múltipla / evaporação do solvente. As partículas foram avaliadas em relação à morfologia, à eficiência de encapsulação, às interações polímero-fármaco, bem como à cinética de liberação do fármaco in vitro . As nanopartículas são esféricas e isoladas, com um diâmetro médio de cerca de 600 nm. O comportamento térmico (DSC) das nanopartículas contendo CEF sugeriu que o fármaco está disperso em um nível molecular dentro do sistema. A eficiência de encapsulação do fármaco no sistema quando a concentração de CEF é 30 mg / mL foi de 5,5%, determinada após a extração de fármaco, através de um método de HPLC validado. Esta baixa eficiência de encapsulação é compreensível, uma vez que a CEF é altamente hidrofílica. Os ensaios in vitro mostraram um perfil de liberação do fármaco a partir das nanopartículas fortemente sustentado e com uma cinética de difusão Fickiana pura.

Nanoparticles containing cefoxitin (CEF) made of D,L-PLA (PLA) were designed by a multiple emulsion/solvent evaporation method. The particles were extensively evaluated in relation to morphology, encapsulation efficiency, drug-polymer interactions as well as in vitro drug release kinetics. Nanoparticles were spherical in shape and isolated, with a mean diameter of about 600 nm. The thermal behaviour (DSC) of CEF-containing nanoparticles suggested that the drug was dispersed at a molecular level within the system. The drug encapsulation efficiency in the system for a CEF concentration of 30 mg/mL was 5.5%, as assessed after the drug extraction, by a validated HPLC method. This low encapsulation efficiency is understandable, since CEF is highly hydrophilic. The in vitro assays showed a strong sustained drug release profile from the nanoparticles with kinetics following pure Fickian diffusion.

Modeling a system of phosphated cross-linked high amylose for controlled drug release. Part 2: physical parameters, cross-linking degrees and drug delivery relationships.

High amylose cross-linked to different degrees with sodium trimetaphosphate by varying base strength (2% or 4%) and contact time (0.5-4h) was evaluated as non-compacted systems for sodium diclophenac controlled release. The physical properties and the performance of these products for sodium diclophenac controlled release from non-compacted systems were related to the structures generated at each cross-linking degree. For samples at 2% until 2h the swelling ability, G' and eta* values increased with the cross-linking degree, because the longer polymer chains became progressively more entangled and linked. This increases water uptake and holding, favoring the swelling and resulting in systems with higher viscosities. Additionally, the increase of cross-linking degree should contribute for a more elastic structure. The shorter chains with more inter-linkages formed at higher cross-linking degrees (2%4h and 4%) make water caption and holding difficult, decreasing the swelling, viscosity and elasticity. For 2% samples, the longer drug release time exhibited for 2%4h sample indicates that the increase of swelling and viscosity contribute for a more sustained drug release, but the mesh size of the polymeric network seems to be determinant for the attachment of drug molecules. For the 4% samples, smaller meshes size should determine less sustained release of drug.

Estimate of the digestibility, assimilability and intestinal permeability of butyltins occurring in wine.

An estimate of the digestibility and assimilability of butyltins occurring in contaminated wines (Port, red and white) was obtained by means of in vitro studies of gastrointestinal digestion. The influence of the wine matrix on the intestinal permeability was explored by studying the accumulation of butyltins in Caco-2 monolayers either when these species are dissolved in buffer only or in the dialysates of digested wines. Some important information about the fate of the butyltin compounds ingested from contaminated wines could be achieved. Only a very small fraction of the ingested DBT and TBT, the two most toxic forms, appear to be able to reach the epithelium as judged by the small dialyzable fraction found (<2%). This is probably independent from the food/drink matrix introducing these contaminants, since the influence of the involved enzymes appear to be dominant, especially for DBT and TBT. Additionally, the intestinal permeability of the three butyltins was also very low, the wine matrix possibly having a hindrance effect in a few cases.

Praziquantel-loaded PLGA nanoparticles: preparation and characterization.

Nanoparticles containing praziquantel made of poly (D,L-lactide-co-glycolide) were designed by an emulsion-solvent evaporation method. Two organic solvents were separately utilized as disperse phase: methylene chloride and ethyl acetate. The size of the particles prepared with the former solvent was bigger than the particles prepared with the latter. The entrapment efficiency was bigger when methylene chloride was used, 79.82% in comparison with 29.27% by using ethyl acetate. DSC and infrared studies showed that no strong chemical interaction between drug and polymer occurred. Release kinetics of praziquantel, used as a model drug, was governed not only by actual drug loading but also by particles size. The higher the drug content and the smaller the particle size resulted in faster drug release.

Estimation of the human intestinal permeability of butyltin species using the Caco-2 cell line model.

The main objective of the work was the setup of the Caco-2 human intestinal cell-line model for the study of the intestinal permeation of monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT). The study was focused in gathering information on (a) the relative permeability of butyltins, (b) their possible permeation routes (paracellular/transcellular) and (c) the eventual interactions between the different butyltins when occurring as a mixture. The presence of basolateral serum protein greatly influenced the permeability, causing a large net clearance, but the apparent permeability (Papp) values were comparable to that of phenolred, suggesting a low in vivo permeability of the butyltins. The found permeability pattern correlates well with the general in vivo toxicity pattern (trialkyltin>dialkyltin>>monoalkyltin). The accumulation pattern (DBT>TBT>MBT) was different from that of permeability and may be an important element regarding the elucidation of some specific strong toxic effects caused by the dialkyltins in several species. The transport of MBT and DBT was found to be dependent on the paracellular route status. An interaction between the butyltin compounds in a mixture was found for the accumulation results (the accumulation was significantly higher for the three compounds when in a mixture). A set of useful information about the butyltin accumulation and transport by the epithelial Caco-2 cell line was, thus, achieved, constituting a starting point for future research on the permeability of butyltins from contaminated food and beverages.

Determination of calcium by atomic absorption spectrophometry applied to the preparation of alginate: chitosan complexes

Polysaccharides, as alginate and chitosan, have been used to obtain modified release dosage forms. Alginate, due to its porperty of building gels during complex formation with calcium ions, allows the building of capsules containing a core constituted by calcium alginate. This work had for objective to determine the appropriate calcium concentration for the preparation of alginate-chitosan capsules, by means of calcium quantification using atomic absorption spectrophotometry. The methodology of calcium quantification was validated through analysis of the limit of detection, precision, accuracy and recovery of the method. The capsules, containing or not the drug, were prepared by the complex coacervation/ionotropic gelification method. Calcium was quantified after samples mineralization and dilution in lantanium solution. The results showed that the amount of calcium incorporated into the capsules depends on the amount of calcium added to the medium, and this ratio increases until the concentration of 1,5% of initial calcium chloride and above this concentration there is a decrease in the proportion of calcium bonded. It was observed that the proportion of calcium that links to the polymer is inversely proportional to the amount of calcium added. The calcium amount incorporated depends on the concentration of the polymeeric dispersions used as well as on the between the two polymers

Microspheres of alginate-chitosan containing isoniazid.

Isoniazid was encapsulated into microspheres of alginate-chitosan by means of a complex coacervation method in an emulsion system. Since the encapsulation of isoniazid tends to be limited by its hydrophilic characteristics, this study proposes its microencapsulation by adsorption. The particles were prepared in three steps: (1) preparation of a W/O emulsion; (2) phase separation; and (3) adsorption of the drug. The isolated particles were placed in a solution of the drug under stirring to allow adsorption. The morphology and particle size were analysed by scanning electron microscopy (SEM). The isoniazid content was determined by extraction in 1 M phosphate buffer pH 7.5 under stirring for 4 h. Finally, the samples were filtered and analysed in an UV/VIS spectrophotometer at 260 nm. In vitro release tests were carried out in 0.05 M phosphate buffer pH 7.5. The results showed that microspheres of alginate-chitosan obtained were of spherical shape. The emulsion used for microparticle formation allows the preparation of particles with a narrow size distribution. The adsorption observed is probably of chemical nature, i.e. there is an ionic interaction between the drug and the surface of the particles.

Characterization of highly sulfated cyclodextrins.

A class of highly sulfated cyclodextrins (HS-CDs) was developed for enantiomeric separation of chiral compounds by capillary electrophoresis (CE). The HS-CDs were produced by a facile single-step direct sulfation of cyclodextrin using sulfur trioxide-trimethylamine complex in dimethylformamide. Characterization of the HS-CDs by electrospray ionization mass spectrometry and by CE using a well-established indirect detection method indicated the species have very narrow heterogeneity in terms of degree of sulfation. Elemental analysis of the HS-alpha-, beta- and gamma-CDs showed that the average sulfate contents were 11, 12, and 13 per CD molecule, respectively. The 13C NMR of HS-CDs is consistent with the structural assignment of nearly complete sulfation at C-6 primary hydroxyl groups and partial sulfation at the C-2 secondary hydroxyls (>70%), while the C-3 hydroxyls remain unsubstituted. Enantiomeric separation by CE using the HS-CDs as chiral selectors showed that HS-alpha-, beta- and gamma-CDs complement each other by exhibiting different chiral selectivities, resulting in resolution of many chiral neutral, acidic and basic compounds of greatly varying structural features. The part of HS-CD that interacts with the guest molecule during complexation and, therefore, the receiving end of the cyclodextrin hydrophobic bucket was surrounded with largely regiospecifically substituted C-2 sulfates and intact C-3 hydroxyls, both at the equatorial positions. Such global regiospecific structural arrangement in HS-CDs provides differential diasteroisomeric complexation is proposed to be the principal contributing factor in the resolving racemates.

Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis.

A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis.

A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS-monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.

Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase.

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.

An intelligent system for the diagnosis of complex images.

An intelligent system suitable to perform a computer aided diagnosis of complex images should have a knowledge base containing all information related both to the images to be interpreted and to their symbolic description. In this paper, a system able to classify unknown medical digital images into four classes is proposed (searched pathology recognized, searched pathology absent, different pathology from the searched one recognized, unknown pathology). A main component of this system is a knowledge base that, startling from information deduced from sample images, can be processed to create synthetic reference models that, in turn, permit the interpretation of real scenes. The system has been tested on digitized plain film of the thorax, in order to perform a computer-aided diagnosis of pneumothorax cases.

Characterization of fluorescent nucleoside triphosphates by capillary electrophoresis with laser-induced fluorescence detection: action of alkaline phosphatase and DNA polymerase.

A method of analysis of fluor-labeled nucleoside triphosphates based on alkaline phosphatase-catalyzed sequential cleavage of phosphate groups with monitoring of all fluorescent species by capillary electrophoresis with laser-induced fluorescence detection is presented. The method allows determination of the purity of the triphosphate samples as well as the relative amounts of the lower phosphate contaminants. The ability of one of the fluor-labeled nucleoside triphosphates to serve as polymerase substrate was verified by labeling DNA restriction fragments by the method of filling recessed 3'-ends using DNA polymerase Klenow fragment.

Acid-catalyzed reductive amination of aldoses with 8-aminopyrene-1,3,6-trisulfonate.

The reductive amination of monosaccharides with 8-aminopyrene-1,3,6-trisulfonate (APTS) in seven different organic acids including the commonly used acetic acid was investigated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. The correlation between the yields of the saccharide-APTS adducts and pKa of the organic acid catalyst is consistent with general acid catalysis of the rate-determining step of the reductive amination reaction. Derivatization in the presence of organic acids of higher strength than acetic acid produced substantially higher yields of APTS-sugar adducts, an effect which is more pronounced for N-acetylamino sugars. Optimum yields were obtained using citric acid as a catalyst. Conversion of a few nanomoles of neutral saccharides to the APTS derivatives is achieved at 75 degrees C in less than 60 min.

Separation of 1-aminopyrene-3,6,8-trisulfonate-labeled asparagine-linked fetuin glycans by capillary gel electrophoresis.

Asparagine-linked glycans of bovine fetuin were separated by capillary gel electrophoresis after enzymatic release (peptide-N-glycosidase F) and labeling via reductive amination by a fluorescent dye, 1-aminopyrene-3,6-8-trisulfonate (APTS). At low separation pH (2.5) only two dominant peaks were observed. Increasing the separation buffer pH to 4.75 resulted in complete separation of two primary doublets and several minor peaks from the fetuin N-linked glycan pool. Two of the four major peaks were spiked with purified individual standards and were identified as trisialylated triantennary structures with different sialylation linkages. The other two larger peaks were postulated to be tetrasialylated triantennary structures, based on calculations considering their corresponding glucose unit (GU) values. Effects of the electrophoretic separation parameters, such as gel concentration, electric field strength and temperature on the migration behavior of the two major doublets of the fetuin glycan pool were also thoroughly examined. Our data suggest that the capillary gel electrophoresis separation of the multisialylated branched oligosaccharides with different linkage isomers, released from bovine fetuin, is fundamentally based on their degree of sialylation and hydrodynamic volumes.

High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate.

High-resolution capillary gel electrophoresis was used for the separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS-saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separated and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown.

Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with laser-induced fluorescence.

A method of analysis of isomers of mono- and oligosaccharides derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) by capillary electrophoresis (CE) in simple buffer systems is presented. The derivatization chemistry is performed at 75 degrees C for 1 h for most mono- and oligosaccharides. Sialyllactose was derivatized at 37 degrees C for 15 h without significant desialylation. Most of the APTS derivatized isomers of mono- and oligosaccharides are resolved in buffers such as acetate (pH 5.0), 3-(N-morpholino)-propanesulfonic acid (pH 7.0), and phosphate (pH 7.4). The mechanism of CE separation using the above buffers is based on the differences in hydrodynamic volume of the derivatized species and is different from that in borate or high pH buffer.