Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system

Influence of the immobilization chemistry on the properties of immobilized b-galactosidases

bstract Ž ) Ž . Neutral b-galactosidases from E. coli and K. lactis were bound to glutaraldehyde-agarose Glut-agarose through Ž . amino groups, and to thiolsulfinate-agarose TSI-agarose through thiol groups. In general, TSI-gels exhibited higher yields Ž . Ž . after immobilization 60-85% than Glut-gels 36-40% . The kinetic parameters of the enzymes bound to TSI-gels Ž . particularly those with lower concentration of active groups were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in Ž . the presence of ethanol, dioxane and acetone 18% vrv . The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents.

Hidrólisis enzimática de lactosa en leche y permeados de lactosuero con beta-galactosidasa (Bacillus circulans) inmovilizada en resinas acrílicas entrampadas en una matriz de alginato

El lactosuero es una importante fuente de nutrientes, ya que contiene un poco más del 25% de las proteínas de la leche, cerca del 8% de la materia grasa y del 95% de la lactosa (Smithers y Copeland, 1997). La alternativa de reaprovechamiento que más se ha explorado en los últimos años es la recuperación de los nutrientes de alta calidad que tiene el lactosuero, mediante la aplicación de procesos de membranas, transformándolo en polvo (Byrne y Fitzpatrick, 2002). En el caso de la ultrafiltración las membranas utilizadas retienen las proteínas del suero y son completamente permeables a lactosa y sales minerales, dando como resultado la obtención de concentrados proteicos (Zall, 1992), mientras que la nanofiltración permite además la retención del componente lactosa del suero (Alkhatim y col., 1998). Así puede encontrarse en algunas industrias lácteas, procesos para obtener suero entero deshidratado, suero desmineralizado, concentrados proteicos y lactosa refinada. No obstante, en la actualidad se siguen descartando grandes cantidades de suero. Aproximadamente el 47% de los 115 millones de toneladas anuales de suero producidas en el mundo se dispone en ríos, lagos y otros cuerpos de agua. Esto representa una significativa pérdida de recursos y causa serios problemas de polución dado que el suero es un contaminante orgánico con altas DBO (40.000-60.000 ppm) y DQO (50.000-80.000 ppm). Más del 90% de esta DBO se debe a la lactosa. Por consiguiente, es importante desarrollar procesos que permitan utilizarlo integralmente con el fin adicional de no contaminar el medio ambiente y de recuperar, con creces, el valor monetario del mismo. El diseño de bioprocesos para el aprovechamiento de la lactosa como recurso, con la generación de productos de mayor valor agregado, ha constituido un objetivo de muchos investigadores en las últimas décadas. Una interesante posibilidad para el aprovechamiento del permeado es la producción de jarabes de lactosa hidrolizada, obteniéndose glucosa y galactosa, dos monosacáridos con mayor poder edulcorante y más solubles que la lactosa (Zhou y Dong-Chen, 2001). Este cambio en las propiedades permite el uso de los jarabes de glucosa galactosa en un gran número de aplicaciones.Fil: Torres, Pedro. Universidad de la República; UruguayFil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Regenhardt, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera". Universidad Nacional del Litoral. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera"; ArgentinaFil: Batista-Viera, Francisco. Universidad de la República; UruguayFil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy

l-Arabinose isomerase is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36.Fil: Torres, Pedro. Universidad de la Republica; UruguayFil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Batista Viera, Francisco. Universidad de la Republica; UruguayFil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentin

Characterization of Galactosyl Derivatives Obtained by Transgalactosylation of Lactose and Different Polyols Using Immobilized beta-Galactosidase from Aspergillus oryzae

The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galacto- side. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl- glycerol, and galactosyl-erythritol by immobilized β-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D (1H and 13C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The β-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.Peer reviewe

Knee ligament injuries: biomechanics comparative study of two suturetechnique in tendon - analysis "in vitro" tendon of bovine

OBJECTIVE: To evaluate and compare the biomechanical behavior of two different suture configurations: "X" and "Loop" in the preparation of tendons for knee ligament reconstruction. METHODS: We used common digital extensor tendons of bovine that can replace the human flexor tendons in experimental studies of traction. In the first group, point "X" suture with Ethibond (r) No. 5 began in the distal graft points transfixing, with spacing of 7.5 mm points to reach 03 cm distal to the beginning of the suture, returning suture in the same manner, transfixing the tendon in open spaces across the suture configuration "X". The second group, the point "Loop" was prepared with the same type Ethibond (r) No. 5 of the needle wire was removed for use only of the wire was mounted in a twofold manner in a single piece forming a needle loop. Started the suture 3 cm from the end of the graft through loops and transfixing points throughout the tendon substance, with spacing between dots of 7.5 mm. RESULT: The Maximum Force of Rupture suture in "Loop" was 444.45 N and the suture in "X" was 407.59 N with statistical significance (p = 0.030). The average Tension obtained at the suture in "Loop" was 27.67 MPa and at the suture in "X" was 25.73 MPa with a statistically significant difference (p = 0.036). The stiffness showed no statistical differences (p = 0.350) at 11.804 N / mm at the point where "Loop" and 11.570 N / mm at the suture "X". CONCLUSION: The suture in "Loop" had a higher biomechanical behavior to the suture "X", considering the Maximum Force and Tension

Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents