Molecular separation by thermosensitive hydrogelmembranes

A new method for separation of molecules of different size is presented. The method is a useful addition to conventional separation methods which depend mainly on gel permeation chromatography using size exclusion. In the new method, hydrogel membranes are used which swelling level can be thermally controlled. In this study, a crosslinked poly(N-isopropylacrylamide¿co-butylmethacrylate 95:5mol%) membrane is used and three solutes of distinct molecular size: two dextrans with molecular weights of 150,000 and 4,400 g/mol respectively and uranine with a molecular weight of 376 g/mol. The swelling of the membranes as function of temperature was measured as well as the influence of the swelling level on the permeability of the three solutes. the influence of the swelling level and the solute size on the permeability was as expected from the free-volume theory. Based on these permeability phenomena, separation was performed in a continuous way by varying the membrane swelling at the appropriate time. A linear relationship between inverse membrane hydration and solute diffusion was found for uranine and dextran (MW=4,400), indicating the validity of the free-volume theory

Heparin release from thermosensitive hydrogels

Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic) comonomers. The incorporation of hydrophobic and hydrophilic comonomers strongly influences the swelling/shrinking behavior of TSH. Upon deswelling, gels containing the hydrophobic comonomer formed a skin-type layer, which acted as a rate controlling membrane, while the hydrophilic comonomer greatly increased gel swelling, relative to NiPAAm. Equilibrium swelling in isotonic PBS and deswelling kinetics of the synthesized gels were examined at various temperatures. The loading of heparin into the different gels was studied as a function of temperature, solution concentration, and gel composition. The release kinetics of heparin was found to be influenced by gel composition and loading temperature; the release correlated with the gel deswelling kinetics. In the case of Ni-PAAm/BMA gel, the release profile of heparin was affected by temperature dependent properties of the skin-type diffusional barrier formed on the gel surface

Release of proteins via ion exchange from albumin-heparin microspheres

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg lysozyme on albumin-heparin microspheres was linear until saturation was abruptly reached,\ud \ud The adsorption isotherms of human lysozyme at low and high ionic strength were typical of adsorption isotherms of proteins on ion exchange gels. The adsorption of human lysozyme on albumin-heparin and albumin microspheres fit the Freundlich equation suggesting heterogeneous binding sites. This was consistent with the proposed multivalent, electrostatic interactions between human lysozyme and negatively charged microspheres. Scatchard plots of the adsorption processes of human lysozyme on albumin-heparin and albumin microspheres suggested negative cooperativity, while positive cooperativity was observed for chicken egg lysozyme adsorption on albumin-heparin microspheres.\ud \ud Human lysozyme loading of albumin-heparin microspheres was 3 times higher than with albumin microspheres, with long term release occurring via an ion exchange mechanism. Apparent diffusion coefficients of 2.1 × 10-1 and 3.9 × 10-11cm2/sec were obtained for the release of human lysozyme from albumin-heparin and albumin microspheres, respectively. The release was found to be independent of diffusion, since the rate determining step was likely an adsorption/desorption processes. An apparent diffusion coefficient of 4.1 × 10-12 cm2/sec was determined for the release of chicken egg lysozyme from albumin-heparin microspheres.\ud \ud Low release of the lysozymes from albumin-heparin microspheres was observed in deionized water, consistent with the proposed ion exchange release mechanism. Overall, albumin-heparin microspheres demonstrated enhanced ion exchange characteristics over albumin microspheres

Release of macromolecules from albumin-heparin microspheres

Hydrophilic microspheres based on albumin-heparin conjugates have been prepared as a macromolecular delivery system. The soluble albumin-heparin conjugate was synthesized and crosslinked in a water-in-oil emulsion with glutaraldehyde to form microspheres in the same manner as for albumin microsphere preparation. The microspheres were characterized in terms of their size and swelling properties. The loading of macromolecules into albumin-heparin microspheres was carried out concurrently and after microsphere preparation. FITC-dextran was applied as a model macromolecule. A higher loading content was achieved when loading was carried out concurrently with microsphere preparation than when loaded subsequently. Prolonged release of FITC-dextran from albumin-heparin microspheres was achieved and attributed to the high molecular weight of the macromolecule. The release of FITC-dextran was modulated by crosslinking density, loading content and the method of drug incorporation. Apparently, the mechanism of FITC-dextran release from albumin-heparin microspheres was dependent on the method of drug incorporation. For release of FITC-dextran from the microspheres, assuming negligible interactions, a diffusion coefficient of 1.7 × 10¿9 cm2/s was determined

Thermosensitive Interpenetrating Polymer Networks:Synthesis, Characterization, and Macromolecular Release

Thermosensitive semiinterpenetrating polymer networks (semi-IPNs) composed of cross-linked poly(N-isopropylacrylamide) (NiPAAm) and linear poly(ether(urethane-urea) (Biomer) were obtained via UV-initiated solution polymerization. The semi-IPNs exhibited negative thermosensitivity, i.e., lower swelling levels with increasing temperature. The incorporation of a relatively small content of Biomer (up to 10 wt %) strongly influenced the mechanical properties, equilibrium swelling, and deswelling kinetics of synthesized networks. The semi-IPNs exhibited greater mechanical strength compared to the cross-linked poly(NiPAAm). Equilibrium swelling levels of the semi-IPNs at low temperatures were markedly decreased due to hydrophobic contribution of Biomer and higher apparent effective cross-linking densities of these networks. The gel collapse point, related to the lower critical solution temperature of poly(NiPAAm), was not affected. The semi-IPNs showed much faster deswelling rates compared to the cross-linked poly(NiPAAm). It was hypothesized that the presence of Biomer prevented the formation of a skin-type layer which normally retards the deswelling process of cross-linked poly(NiPAAm). Loading and release of heparin, a model macromolecule, was studied as a function of temperature and Biomer content in semi-IPNs. The partition coefficients of heparin within the networks decreased with increasing temperature and Biomer content. Similarly, a linear relationship between partition coefficients and equilibrium swelling in loading solutions was found for all synthesized networks. Heparin release profiles correlated with deswelling kinetics of crosslinked poly(NiPAAm) and NiPAAm/Biomer semi-IPNs. Release profiles were in agreement with the proposed mechanism of solute release from swollen thermosensitive gels

Preparation and characterization of microspheres of albumin-heparin conjugates

Albumin-heparin microspheres have been prepared as a new drug carrier. A soluble albumin-heparin conjugate was synthesized by forming amide bonds between human serum albumin and heparin. After purification the albumin-heparin conjugate was crosslinked in a water-in-oil emulsion to form albumin-heparin microspheres. The composition of the conjugate was determined by amino acid analysis. The swelling properties of albumin-heparin microspheres were investigated as a function of pH and ionic strength and compared with albumin microspheres. Albumin-heparin and albumin microspheres exhibited stimuli-sensitive swelling. Both microsphere systems exhibited low swelling at low pH and high swelling at higher pH caused by ionization of amino acids of serum albumin. The swelling of albumin-heparin microspheres was more sensitive toward ionic strength than that of albumin microspheres. This was due to the greater negative charge of the albumin-heparin microspheres. Surfaces of albumin-heparin and albumin microspheres were characterized by ESCA, contact angle measurements, electrophoresis, and scanning electron microscopy. Surface analysis indicated the presence of heparin at the albumin-heparin microsphere/water interface

Identification of DNA methylation changes associated with human gastric cancer

<p>Abstract</p> <p>Background</p> <p>Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult.</p> <p>Methods</p> <p>We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay) in combination with a genome analyzer and a new normalization algorithm.</p> <p>Results</p> <p>We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs), transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the <it>HOX </it>and histone gene families. We also discovered long-range epigenetic silencing (LRES) regions in gastric cancer tissue and identified several hypermethylated genes (<it>MDM2</it>, <it>DYRK2</it>, and <it>LYZ</it>) within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue.</p> <p>Conclusions</p> <p>Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.</p

Importância dos Saca-Rabos (Herpestes Ichneumon) como Reservatório de Mycobacterium avium subsp. paratuberculosis. Deteção por Técnicas Tradicionais e Moleculares

Poster apresentado nas IV Jornadas de Genética, realizadas na UTAD, Vila Real, nos dias 1,2 e 3 de Março de 2012.Os saca-rabos (Herpestes ichneumon) também conhecidos por mangustos, são carnívoros diurnos selvagens que juntamente com a geneta (Genetta genetta) representam os exemplares da família Viverridae em Portugal. É uma espécie cinegética de caça menor que se alimenta de coelhos, roedores, aves, cobras, insectos e ovos. Neste estudo colheram-se amostras de 8 animais mortos por atropelamento e em ações de controlo de predadores, durante os anos de 2010 e 2011, nos concelhos de Idanha-a-Nova e Penamacor do distrito de Castelo Branco. As amostras colhidas foram fígado, pulmão, baço, intestino, rim, gânglio mesentérico, retrofaríngeo, mediastínico, amígdalas e fezes. As amostras foram submetidas à técnica de PCR e a cultura microbiológica em meios específicos. Em três saca-rabos (37,5%) detectou-se Mycobacterium avium subsp. paratuberculosis (Map) através da técnica de biologia molecular. Dois eram machos e um era fêmea. Map foi confirmado também em cultura nos dois machos. Sete saca-rabos (87,5%) apresentaram bactérias álcool-ácido resistentes compatíveis com Map em esfregaços de diferentes tecidos, quando corados pelo método de Ziehl-Neelsen. Estes resultados preliminares confirmam os saca-rabos como reservatório de Map no nosso país. Atualmente, estão a ser desenvolvidos mais estudos para a avaliação dos saca-rabos na dinâmica da infeção de Map em mamíferos selvagens

Successful Treatment of Stereotactic Body Radiation Therapy Combined with Transarterial Chemolipiodolization for Hepatocellular Carcinoma with Biliary Obstruction

Conventional radiation therapy (RT) is a widely recognized treatment for hepatocellular carcinoma (HCC). However, conventional RT plays only a limited role in HCC treatment because of its low efficacy and the low tolerance of the liver for this modality. Stereotactic body radiation therapy (SBRT) was recently developed and represents the most advanced radiation therapy technique currently available. It can deliver a high dose in a short time to well-defined hepatic tumors, with rapid dose fall-off gradients. We believe that SBRT with transarterial chemolipiodolization (TACL) may prove promising as a combined treatment modality for HCC due to its precision and relative safety. Here we present a case of successful treatment of advanced HCC with obstructive jaundice using this combined modality