Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Caracterización del polirribosilribitol fosfato por cromatografía de exclusión molecular de alta eficacia con detección ultravioleta / Characterization of polyribosylribitol phosphate by high efficiency size exclusion chromatography with ultraviolet detection

Haemophilus influenzae tipo b es un importante patógeno del hombre causante de varias de las enfermedades invasivas en niños menores de cinco años, contra el cual fueron autorizadas las vacunas glicoconjugadas a partir del polirribosilribitol fosfato. Quimi-Hib® es la primera y única vacuna contra este patógeno que utiliza el polisacárido obtenido por síntesis química. El Ingrediente Farmacéutico Activo es producido por el Centro de Ingeniería Genética y Biotecnología y se obtiene a partir de su conjugación al toxoide tetánico. En el presente reporte se hizo una caracterización del polirribosilribitol fosfato mediante la técnica de cromatografía de exclusión molecular de alta eficacia con detección ultravioleta a 215 nm. En el estudio se evaluaron tres lotes y se determinó el perfil de elución en una columna SuperdexTM 75 10/300 GL Increase con un porciento de pureza de 77,42 ± 8,97 y una masa molar promedio de 7.381 Da ± 210,93. La principal impureza presente en el polirribosilribitol fosfato es el dimetilsulfóxido, disolvente utilizado en la reacción de activación con el éster N-hidroxisuccinimidilo del ácido β-maleimidopropiónico. El polirribosilribitol fosfato se purificó por filtración con un Amicon Ultra-15 de 2.000 Da hasta una pureza de 99,1 por ciento y se conjugó al toxoide tetánico. El rendimiento de la reacción de conjugación con el polisacárido purificado fue de 30,0 por ciento 1,77 el cual no muestra diferencias significativas con el control que fue 33,7 por ciento ± 3,57 demostrándose que el dimetilsulfóxido no afecta el desempeño de la reacción de conjugación(AU)

Haemophilus influenzae type b is an important human pathogen causing some invasive diseases in children less than five years of age. Glycoconjugate vaccines based on polyribosylribitol phosphate have been licensed against this bacterium. Quimi-Hib® is the first and only vaccine against this pathogen using the chemically synthesized polysaccharide. The Active Pharmaceutical Ingredient is produced by the Center for Genetic Engineering and Biotechnology and is obtained from its conjugation to tetanus toxoid. In the present report a characterization of polyribosylribitol phosphate was performed by high performance molecular exclusion chromatography with ultraviolet detection at 215 nm. Three batches were evaluated in the study and the elution profile was determined on a SuperdexTM 75 10/300 GL Increase column with a purity percentage of 77.42 ± 8.97 and an average molecular weight of 7,381 Da ± 210.93. The main impurity present in polyribosylribitol phosphate was dimethylsulfoxide, the solvent used in the activation reaction with N-hydroxysuccinimidyl ester of β-maleimidopropionic acid. Polyribosylribitol phosphate was purified by filtration using a 2,000 Da cut-off Amicon Ultra-15 to a purity of 99.1 percent and conjugated to tetanus toxoid. The yield of the conjugation reaction with the purified polysaccharide was 30.0 percent ± 1.77 which shows no significant difference with the control which was 33.7 percent ± 3.57 demonstrating that dimethylsulfoxide does not affect the performance of the conjugation reaction(AU)

Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.

Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography / 生物工程学报

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.

Purification and characterization of two PR-10 protein isoforms from the crude drug of Angelica sinensis / 生物工程学报

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.

Desenvolvimento de vesículas poliméricas de poli(etileno glicol)-b-poli(ε-caprolactona) (PEG-PCL) para veiculação de L-asparaginase / Development of polyethylene glycol-polycaprolactone polymer vesicles for L-Asparaginase

A L-Asparaginase (ASNase) é um importante agente quimioterapêutico utilizado para o tratamento da leucemia linfoblástica aguda (ALL) há mais de 40 anos. No entanto, devido à origem biológica da ASNase, enzima produzida por Escherichia coli, problemas como a imunogenicidade e baixa meia vida-plasmática devem ser considerados. Com o objetivo de minimizar essas desvantagens, várias ASNases homólogas bem como formulações de ASNase de E. coli foram investigadas. Nenhuma das formulações desenvolvidas, entretanto, foi capaz de resolver definitivamente esses problemas associados à sua origem. Nesse sentido, considerando os recentes avanços na ciência de polímeros com a possibilidade do obtenção de vesículas poliméricas usando copolímeros, este trabalho concentrou-se no desenvolvimento de polimerossomos de poli(etileno glicol)-b-poli(ε-caprolactona) (PEG-PCL) para encapsular a ASNase. Diversas condições experimentais foram investigadas e, ao final, os polimerossomos foram produzidos pela técnica de hidratação do filme polimérico utilizando a centrifugação como técnica de pós-filme para remoção de copolímero precipitado, produzindo assim vesículas polímericas de 120 a 200nm com PDI de aproximadamente 0,250. A eficiência de encapsulação da ASNase, utilizando as metodologias de centrifugação ou cromatografia de exclusão molecular, revelou taxas de encapsulação de 20-25% e 1 a 7%, repectivamente. Esses resultados apontam a importância de se determinar a eficiência de encapsulação por cromatografia de exclusão molecular ou método direto no caso de nanoestruturas auto-agregadas formadas por copolímeros, devido a valores superestimados com o emprego da centrifugação. Ainda que estudos complementares se façam necessários para liberação da enzima encapsulada ou penetração da L-asparagina nas vesículas, nossos resultados demonstram o potencial de polimerossomos para veiculação de ASNase, bem como de outras proteínas terapêuticas

L-Asparaginase (ASNase) is an important chemotherapeutic agent used for the treatment of acute lymphoblastic leukemia (ALL) for more than 40 years. However, due to the biological origin of ASNase (produced by Escherichia coli) some drawbacks such as immunogenicity and low plasma half life are present. In order to minimize the disadvantages, several ASNases proteoforms and formulations of E. coli ASNase were investigated. However, none of this formulations completely solved the main drawbacks of ASNase. In this sense, considering the recents advances in polymers science with the possibility to develop polymeric vesicles using copolymers, this work aimed at the development of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) vesicles to encapsulate ASNase. Different experimental conditions were investigated and, the final polymersomes formulation was prepared by film hydratation using centrifugation as a post-film technique to remove the bulky coplymer. Polymeric vesicles of 120 to 200nm with PDI of approximately, 0.250 were obtained. The encapsulation efficiency of ASNase was determined indirectly by centrifugation and directly by size exclusion chromatography, resulting in encapsulation rates of 20-25% and 1 to 7%, respectively. These results indicate the importance of determining the efficiency of encapsulation by size exclusion chromatography or direct method in the case of self-aggregated nanostructures formed by copolymers, due to values overestimated with the use of centrifugation. Our results point to the potential of polymersomes for ASNase delivery, as well as other therapeutic proteins. Nonetheless, complimentary studies are still necessary for ASNase release or L-asparagine penetration into the vesicles

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration / Avaliação da qualidade do bevacizumabe (Avastin®) fracionado em frascos de vidro de dose única para administração intravítrea

ABSTRACT Purpose: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. Methods: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). Results: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. Conclusion: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

RESUMO Objetivos: Avastin® (bevacizumabe) é um anticorpo monoclonal inibidor do fator de crescimento endotelial de vasos (VEGF) utilizado "off-label" por meio de administração intravítrea para o tratamento de doenças oculares. A sua aplicação clínica associada ao custo-benefício do medicamento gerou uma demanda para seu fracionamento em frascos de dose única para utilização pela via intraocular. No entanto, a segurança do fracionamento do anticorpo em frascos de dose única ainda é alvo de discussão. Neste trabalho, a estabilidade e a eficácia do Avastin® fracionado em frascos ou ampolas de vidro de dose unitária por farmácias de manipulação do mercado foram avaliadas. Métodos: As técnicas de eletroforese em gel de poliacrilamida (PAGE), cromatografia por exclusão de tamanho (SEC), espalhamento dinâmico da luz (DLS) e turbidimetria foram empregadas para avaliar a formação de agregados de diferentes tamanhos. Alterações na atividade biológica do bevacizumabe foram estudadas utilizando ELISA. Resultados: Amostras referência e do bevacizumabe fracionado apresentaram resultados semelhantes quando analisado por gel de poliacrilamida. Por cromatografia por exclusão de tamanho, um pequeno aumento na quantidade de agregados de alta massa molar seguido de uma redução nos monômeros do bevacizumabe foram observados para as amostras das três farmácias de manipulação quando comparado ao referência. A comparação dos cromatogramas mostrou uma quantidade de redução do monômero inferior a 1% para todas as amostras fracionadas. Por espalhamento dinâmico da luz e turbidimetria, não foram detectados agregados de proteína na faixa de tamanho de micrômetro e nanômetro. No ensaio de eficácia, o bevacizumabe fracionado preservou sua função biológica pois apresentou menos de 3% de perda na capacidade de ligação ao VEGF quando comparado ao referência. Conclusão: Este estudo sugere que o bevacizumabe se mantem estável após fracionamento em ampolas e frascos de vidro de dose unitária pois não foram observadas agregação e/ou fragmentação de proteínas e perda de atividade biológica em quan tidades significativas.

Preparation and antioxidant activity detection of collagen peptide from Cirrhinus molitorella skin / 生物工程学报

In order to prepare antioxidant peptide through hydrolyzing low-value protein resources with bacterial extracellular proteases and to discover novel proteases, crude extracellular protease from Pseudoalteromonas sp. SHK1-2 was obtained through fermentation which was used to hydrolyze collagen extracted from Cirrhinus molitorella skin. Small peptide fraction was isolated from hydrolysate by ultrafiltration and Sephadex LH-20 size exclusion chromatography and showed 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity (35.6%±7%), oxygen radical absorbance capacity and inhibition of DNA oxidation damage. The molecule weight was 776.2 Da, and amino acid sequence was Thr-Ala-Gly-His-Pro- Gly-Thr-His through liquid chromatography mass spectrum. Our findings suggest that peptide obtained from low-value protein of fish waste by hydrolysis with bacterial protease has antioxidant activity.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

Copper induction and differential expression of laccase in Aspergillus flavus

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

Resultados de dos encuestas dirigidas a inmigrantes y nativos del sureste español: salud, uso de servicios y necesidad de asistencia médica / Results of two surveys of immigrants and natives in Southeast Spain: health, use of services, and need for medical assistance

Objetivo. Comparar la salud, uso de servicios sanitarios y necesidad insatisfecha de atención médica (NIAM) entre inmigrantes y nativos del sureste español. Material y métodos. Estudio transversal de dos muestras representativas de población: inmigrante (n=1150) y nativa (n=1303; Encuesta Nacional de Salud). Se creó una única base de datos con ponderación específica para cada muestra y se estimaron razones de prevalencia (RP) mediante regresión multivariante. Resultados. Marroquíes, ecuatorianos y europeos del este (EE) declararon peor salud que los nativos (RPs [IC95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] y 1.44 [1.08-1.93], respectivamente). Los inmigrantes hicieron mayor uso de las urgencias (excepto EE) y consumieron menos fármacos. Los marroquíes mostraron la mayor diferencia en la frecuencia de NIAM (RP [IC95%]: 12.20 [5.25-28.37]), principalmente por razones laborales (46%). Conclusiones. La salud y el uso de servicios sanitarios difirieron significativamente entre inmigrantes y nativos. Destaca la NIAM alta en marroquíes por causa laboral.

Objective. To compare the self-perceived health, use of health services and unmet need for health care (UNHC) among immigrants and native populations of Southeast Spain. Materials and methods. Cross-sectional study of two representative samples of 1150 immigrants, and 1303 native participants from the National Health Survey. A single database was created with specific weights for each sample, and prevalence ratios (PR) were estimated by multivariate regression. Results. Moroccans, Ecuadorians and Eastern Europeans (EE) reported poorer health than the native population (PRs [CI95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] and 1.44 [1.08-1.93], respectively). Immigrants made greater use of emergencies that natives (except for EE) and had lower use of medication. Moroccan showed the greatest difference in the frequency of UNHC (PR [CI95%]:12.20 [5.25 - 28.37]), mainly because of working limitations (46%). Conclusions. The health status and use of health services among immigrants differ significantly from those of natives. Results highlight the higher frequency of UNHC among immigrants, especially high in Moroccans.

In the final analysis, are we a consumer society or not? Implications for health / Afinal, somos ou não somos uma sociedade de consumo? Consequências para a saúde

In this paper, the question of Brazil's insertion today as a country with the characteristics of modern consumer societies is discussed, focusing on the commercialization of the health sector, the segmentation of the health system and the contradictions of the rights to health care in the social context in question. Some research data on these issues broadcast in the National News Bulletins of Globo TV during the year of 2012 are presented, in which the high technology private hospital as a consumer icon, the underfunding of the public health system and the rejection of a poor and deprived Unified Health System are analyzed.

Discute-se aqui a nossa inserção como país, hoje, nas sociedades de consumo características da modernidade, enfocando a mercantilização na área da saúde, a segmentação do sistema de saúde e as contradições do direito à saúde no contexto social em questão. São apresentados dados de pesquisa sobre o tema no Jornal Nacional da Rede Globo de Televisão, durante o ano de 2012, na qual se analisa o hospital privado de alto padrão tecnológico como ícone de consumo, o subfinanciamento do sistema público de saúde e a rejeição de um Sistema Único de Saúde pobre e carente.

Characterization of pumpkin polysaccharides and protective effects on streptozotocin-damaged islet cells / 中国天然药物

The polysaccharides from pumpkin fruit (PP) were obtained and purified by hot-water extraction, anion-exchange chromatography, and gel column chromatography. The physicochemical properties of PP were determined by gel filtration chromatography, gas chromatography, fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Results indicated that the molecular weight of PP was about 23 kDa and PP was composed of D-Arabinose, D-Mannose, D-Glucose, and D-Galactose with a molar ratio of 1 : 7.79 : 70.32 : 7.05. FTIR and NMR spectra indicated that PP was the polysaccharide containing pyranose ring. Additionally, PP protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO. The experiment of reverse transcriptase-polymerase chain reaction further proved that PP inhibited apoptosis via modulating the expression of Bax/Bcl-2 in STZ-damaged islet cells. In conclusion, PP could be explored as a novel agent for the treatment of diabetes mellitus.

Listeriosis en México: importancia clínica y epidemiológica / Listeriosis in Mexico: Clinical and epidemiological importance

La listeriosis es una enfermedad transmitida por alimentos (ETA) y ocasionada por Listeria monocytogenes. La importancia de ésta se debe a su impacto clínico, la alta tasa de mortalidad y el efecto económico derivado de los brotes asociados con el consumo de alimentos. En México, las fallas en los sistemas de vigilancia epidemiológicos son causa de información imprecisa sobre la incidencia de la listeriosis y sobre su caracterización como ETA. En este trabajo se presentan datos referentes a la presencia de la bacteria en alimentos, reportes de casos de la enfermedad y patologías relacionadas con infección por L. monocytogenes. La falta de datos exactos sobre la importancia de esta bacteria plantea la necesidad de concientizar a las instancias correspondientes para definir estrategias de búsqueda intencionada de L. monocytogenes en alimentos y de la recopilación de información clínica precisa que permita conocer la importancia clínica y epidemiológica de la listeriosis en México.

Listeriosis is caused by Listeria monocytogenes, an important food-borne disease due to its clinical forms, high mortality rate, and the economic impact in both clinical and food production industries. In Mexico, the lack of epidemiological surveillance systems leads to the need of accurate data on the incidence of listeriosis and its association with food-borne disease. In this paper, we present data about the presence of this bacterium in food, reports related to clinical cases of listeriosis, and information of diseases in which L. monocytogenes may be involved. However, in most of these cases the etiology was not established. Given this, there's a need to inform and warn the appropriate entities, to define strategies for the mandatory search of L. monocytogenes through the whole food production chain and clinical suspects, for the epidemiological importance and control of listeriosis in Mexico.

Necessidades e preocupações dos pais em diferentes etapas do ciclo vital / Parents' needs and concerns at different stages of the life cycle / Necesidades y preocupaciones de los padres en diferentes etapas del ciclo vital

Objetivo: identificar as necessidades e as preocupações prioritárias, manifestadas pelos pais no desempenho do seu papel, em três etapas do ciclo vital: adolescência, idade produtiva e idade madura. Metodologia: estudo exploratório com abordagem qualitativa, desenvolvido com quatorze pais residentes em um município no extremo sul do Brasil. Os dados foram coletados entre maio e agosto de 2011, por meio de entrevista em profundidade. Através da técnica da análise textual discursiva e da matriz construída com base na teoria bioecológica de Bronfenbrenner, foram construídas três categorias: Necessidades/preocupações do pai, geradas em sua relação com o mundo do trabalho; Necessidades/preocupações que emergem da relação de cuidado com os filhos e Preocupações dos pais com relação ao futuro dos filhos. Conclusão: identificou-se que a preocupação com o futuro dos filhos foi apontada por pais de todas as faixas-etárias investigadas. .

Objective: this study aimed to identify priority needs and concerns expressed by fathers in the performance of their role in three stages of the life cycle: adolescence, productive age, and mature age. Methodology: this is an exploratory study with a qualitative approach, conducted with fourteen fathers residing in a municipality in the extreme south of Brazil. The data were collected between May and August 2011 by means of the in-depth interview. Through the technique of written discourse analysis and the array built upon Bronfenbrenner's bioecological theory, we obtained three categories: fathers' needs/concerns, generated in their relationship with the world of work; needs/concerns that emerged from the relationship of care with the children; and fathers' concerns about the future of the children. Conclusions: we identified that the concern with the future of the children was pointed out by fathers of all age groups investigated. .

Objetivo: identificar las necesidades y preocupaciones prioritarias, manifestadas por los padres en el desempeño de su función, en tres etapas del ciclo de vida: adolescencia, edad productiva y edad madura. Metodología: estudio exploratorio con abordaje cualitativo, desarrollado con catorce padres residentes en un municipio en el extremo sur de Brasil. Los datos fueran colectados entre mayo y agosto de 2011, a través de entrevistas en profundidad. A través de la técnica de análisis textual y discursiva e de la matriz construida basada en la teoria bioecologica de Bronfenbrenner, fueran construidas tres categorías: Necesidades/ preocupaciones de lo padre, generado en suya relación con el mundo de lo trabajo; Necesidades/preocupaciones que emergen de la relación de cuidado con hijos e preocupaciones de los padres con lo futuro de los hijos. Conclusión: Se identifico que la preocupación con el futuro de los hijos fue apuntado por los padres de todas las edades averiguadas. .

Medication adherence and quality of life among the elderly with diabetic retinopathy / Adesão medicamentosa e qualidade de vida em idosos com retinopatia diabética / Adhesión a la medicación y calidad de vida en ancianos con retinopatía diabética

METHOD: one hundred (n=100) elderly outpatients with diabetic retinopathy taking antihypertensives and/or oral antidiabetics/insulin were interviewed. Adherence was evaluated by the adherence proportion and its association with the care taken in administrating medications and by the Morisky Scale. The National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) was used to evaluate HRQoL. RESULTS: most (58%) reported the use of 80% or more of the prescribed dose and care in utilizing the medication. The item "stopping the drug when experiencing an adverse event", from the Morisky Scale, explained 12.8% and 13.5% of the variability of adherence proportion to antihypertensives and oral antidiabetics/insulin, respectively. CONCLUSION: there was better HRQoL in the Color Vision, Driving and Social Functioning domains of the NEI VFQ-25. Individuals with lower scores on the NEI VFQ-25 and higher scores on the Morisky Scale presented greater chance to be nonadherent to the pharmacological treatment of diabetes and hypertension. .

OBJETIVO: investigar os fatores relacionados à adesão medicamentosa e sua relação com a qualidade de vida relacionada à saúde em idosos com retinopatia diabética. MÉTODO: foram entrevistados 100 idosos, em acompanhamento ambulatorial, em uso de anti-hipertensivos e/ou antidiabéticos orais/insulina. A adesão foi avaliada pela proporção de adesão e sua associação com os cuidados no uso dos medicamentos e pela Escala de Morisky. O National Eye Institute Visual Funcioning Questionnaire foi utilizado para avaliar a qualidade de vida relacionada à saúde. RESULTADOS: A maioria (58%) relatou o uso de 80% ou mais das doses prescritas e os cuidados na tomada dos medicamentos. O item "interromper o uso dos medicamentos por se sentir pior", da Escala de Morisky, explicou 12,8 e 13,5% da variabilidade da proporção de adesão aos anti-hipertensivos e aos antidiabéticos orais/insulina, respectivamente. CONCLUSÃO: observou-se melhor qualidade de vida relacionada à saúde nos domínios visão de cores, dirigir automóvel e apectos sociais do National Eye Institute Visual Funcioning Questionnaire. Indivíduos com menor pontuação na National Eye Institute Visual Funcioning Questionnaire e maiores escores na Escala de Morisky apresentaram maiores chances de serem não aderentes aos medicamentos do diabetes e da hipertensão arterial. .

OBJETIVO: investigar los factores relacionados a la adhesión a la medicación y su relación con la Calidad de Vida Relacionada a la Salud (CVRS) de ancianos con retinopatía diabética. MÉTODO: fueron entrevistados cien (n=100) pacientes ancianos de ambulatorio con retinopatía diabética que toman medicamentos antihipertensivos y/o antidiabéticos orales/insulina. La adhesión fue evaluada mediante la proporción de adhesión y su asociación con el cuidado tomado en la administración de medicamentos y mediante la Escala de Morisky. El National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) fue usado para evaluar la CVRS. RESULTADOS: la mayoría (58%) relató el uso de 80% o más de la dosis prescrita y cuidado con el uso de la medicación. El ítem "suspender la droga cuando vivencia un evento adverso", de la Escala de Morisky, explicó 12.8% y 13.5% de la variabilidad en la proporción de adhesión a los antihipertensivos y antidiabéticos orales/insulina, respectivamente. CONCUSIÓN: fue encontrada mejor CVRS en los dominios de Visión Cromática, Dirección y Funcionamiento Social del NEI VFQ-25. Individuos con puntuaciones menores en el NEI VFQ-25 y puntuaciones mayores en la Escala de Morisky revelaron mayor chance de no adhesión al tratamiento farmacológico de la diabetes y hipertensión. .

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.