Nanobiosensores y salud animal.

Los biosensores son dispositivos cuantitativos, semicuantitativos o analíticos, que contienen una biomolécula sensora capaz de convertir una señal biológica en una señal óptica o electroquímica. En los últimos años las nanopartículas de oro se han presentado como una interesante alternativa para la el biosensado de analitos debido a sus propiedades ópticas

Green Production of Cladribine by Using Immobilized 20-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 20-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehydeactivated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5–9) and temperature (30–60 C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.Centro de Investigación y Desarrollo en Ciencias Aplicada

How reliably can algorithms identify eosinophilic asthma phenotypes using non-invasive biomarkers?

Asthma is a heterogeneous respiratory disease that encompasses different inflammatory and functional endophenotypes. Many non-invasive biomarkers has been investigated to its pathobiology. Heany et al proposed a clinical algorithm that classifies severe asthmatic patients into likely-eosinophilic phenotypes, based on accessible biomarkers: PBE, current treatment, FeNO, presence of nasal polyps (NP) and age of onset.We assessed the concordance between the algorithm proposed by Heany et al. with sputum examination, the gold standard, in 145 asthmatic patients of the MEGA cohort with varying grades of severity.No correlation was found between both classifications 0.025 (CI = 0.013-0.037). Moreover, no relationship was found between sputum eosinophilia and peripheral blood eosinophilia count in the total studied population.In conclusion, our results suggest that grouping the biomarkers proposed by Heany et al. are insufficient to diagnose eosinophilic phenotypes in asthmatic patients. Sputum analysis remains the gold standard to assess airway inflammation.© 2022 The Authors. Clinical and Translational Allergy published by John Wiley & Sons Ltd on behalf of European Academy of Allergy and Clinical Immunology

Peripheral T-cell lymphoma: Molecular profiling recognizes subclasses and identifies prognostic markers

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL

Coinmovilización y estabilización de oxidasas y catalasas : aplicaciones biotecnológicas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-04-2005Oxidases have major bioiechnological interest because they are aMe to catalyze under mild d o n mndions. very selecWe oxidations of otganic mpounds by using molealar oxygen as a unique werall oxidmng agent. However, one ofthe main d-s of the industrial use of oxidases is the famiaüon of hydmgen peroxide as a product. mis mpound prwnotes very fast enzyme inactivations by non speafic oxidaüons of soma sansitive residues of the protein or reacüon produds that pmmote ihe decrease in the final yield and the presenca of undesired by-produds in the final reacoOn mixlures. The quantitaüve oxidation of organic wmpounds catalyzed by oxidases thus requires a very careful design of the biocatalyst. In this Thesis we pmpose the coimmabiliin of oxidases with catalases to eüminab the hydrogen peroxide in situ ifu befwe can promote these undesired slde readhns. In o&r to aecompüsh thk main objedve we prepared a wide vaiieiy of immobili preparaüons of catalases ímm different so- (hom bovine l i i r (BLC). Aspefgiiius niger, Mcmwccus /ysodeMcus, and Themus thennqphilus) using different physim chemical methodologies whidi aimed the muiüpont comknt attachment of the enzyme on the suppMt In al1 cases these emymes were stabaPed by Mmobilaation @y a 13.30, 3 and 50 fdd fador for ea& enzyme respecovely). In addiioon. iwo model oi0dases (d-aminoacid oxidase @Man)d glucose oxidase (GOX)) were also immobillred and successMly s t a b i l i by adsorption on calionic mattixes and fucther crosslinking with glutaraldehyde. The immobñized preparaüon of DAAO was M00 times more stabk than the solubte enzyme whereas íhe síablbüon fa& achieved with GOX (a vecy robust enzyme) was 96 with resped io its soluble munterpart. CoimnobiEzed preparaüons of oddasas M BLC were successruily appld to three different biobansfomiaoons: the preparaoai ofadadipyl7- aminocephelosporanic acid from Caphaloymrin C using a DAAOBLC derivative, ihe m r a t i o n of gluconic acid using a GOXBLC -ve and the preparaüon of frudooliiharides from sacamse using a frudosilbansfefase Qsr) derivaove. in whikh the presence of GOX is required due glucose inhibioon of FST. Fw this last purpose, a highly staaüzed derivabLe of FST was also prepared and optimked by adsorpüon on cationic matii%es and further aosslim wiih glutaraldehyde. In aH ihe biiransfomaüons assayed, the effiaency of the oxidaselcatalase mupling was pmved by different me-: by disappearance of a bypmdud fonned by hydrogen pero>Ode (as in the case of a-cetoadipyl7- aminocaphaksporani aQd preparakm), avddance of the inhibihn eííed on ihe FST actMty in ihe preparation of FOS. w elihation of GOX ina@Müon seen as ihe capaQty of completely oxjdize a 100mM glumse soluüon. Aü the coimnobiiked derhtks pwant very good perspedives in the eiuyme technology field due io their good stabEty proparties achieved by an apomi i m m o b ü i protocol and the~derivedímmthe~~mthcatalesewNdinotonlyimpmvssthestabiEtyofthe oxidase decivaove bit also in rpome cases make possible the 7 M

Glutaraldehyde-mediated protein immobilization

In this chapter, we describe different approaches for the utilization of glutaraldehyde in protein immobilization. First, we focus on the covalent attachment of proteins to glutaraldehyde-activated matrixes. We describe conditions for the synthesis of such supports and provide an example of the immobilization and stabilization of fructosyltransferase. We also describe how glutaraldehyde may be used for the cross-linking of protein–protein aggregates and protein adsorbed onto amino-activated matrixes. In these cases, glutaraldehyde bridges either two lysine groups from different proteic molecules or a lysine from the protein structure and an amine group from the support. Examples of cross-linking are given for the immobilization of DAAO on different amino-activated supports.Peer Reviewe

Hydrolysis of tannic acid catalyzed by immobilized-stabilized derivatives of tannase from Lactobacillus plantarum

A recombinant tannase from Lactobacillus plantarum, overexpressed in Escherichia coli, was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditionsThis work was supported by Grants AGL2008-01052, AGL-2009-07625, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD(CICYT),RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). J.A.C. is the recipient of a predoctoral fellowship from the I3P-CSIC Program and FPI-MEC, and G.F.-L. and L.B. are recipients of Ramon y Cajal postdoctoral contracts.Peer reviewe

Hydrolysis of fish oil by lipases immobilized inside porous supports

A new assay was designed to measure the release of omega-3 acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] from the hydrolysis of sardine oil by lipases immobilized inside porous supports. A biphasic system was used containing the fish oil dissolved in the organic phase and the immobilized lipase suspended in the aqueous phase. The assay was optimized by using a very active derivative of Rhizomucor miehei lipase (RML) adsorbed onto octyl-Sepharose. Standard reaction conditions were: (a) an organic phase composed by 30/70 (v:v) of oil in cyclohexane, (b) an aqueous phase containing 50 mM methyl-cyclodextrin in 10 mM Tris buffer at pH 7.0. The whole reaction system was incubated at 25 °C. Under these conditions, up to 2% of the oil is partitioned into the aqueous phase and most of the 95% of released acids were partitioned into the organic phase. The organic phase was analyzed by RP-HPLC (UV detection at 215 nm) and even very low concentrations (e.g., 0.05 mM) of released omega-3 fatty acid could be detected with a precision higher than 99%. Three different lipases adsorbed on octyl-Sepharose were compared: Candida antarctica lipase-fraction B (CALB), Thermomyces lanuginosa lipase (TLL) and RML. The three enzyme derivatives were very active. However, most active and selective towards polyunsaturated fatty acids (PUFA) versus oleic plus palmitic acids (a fourfold factor) was CALB. On the other hand, the most selective derivatives towards EPA versus DHA (a 4.5-fold factor) were TLL and RML derivatives.Peer Reviewe

Jameos : publicación del CEP de Lanzarote

Mayo de 2015, en la sala de exposiciones de Arrecife, el CEP de Lanzarote proyecta, dentro de la semana de arte docente, el documental “Five days to dance”. Dos maestras se deciden a verlo. Setenta minutos donde se narra la experiencia de una pareja de bailarines y coreógrafos que asisten a un instituto en San Sebastián para desarrollar un proyecto: bailar y subirse a un escenario en cinco días. El reto es enorme pero la satisfacción al ver el proceso y el resultado es aún mayor. La danza es mucho más que moverse, es sentimiento, expresión, es mirar al interior de cada uno y conectar con el de al lado. Es contacto, dedicación, disciplina y pasión. Transmitir esto al alumnado adolescente de un instituto y ver lo que son capaces de crear es maravilloso. La idea que surge a partir de este visionado es hacer un proyecto de danza en el CEIP Los Geranios. El siguiente paso fue darle forma, buscar los objetivos y relacionarlos con el currículo. Este proyecto se relaciona directamente con los objetivos que aparecen en el artículo 7 del Real Decreto 126/2014, de 28 de febrero, por el que se establece el currículo básico de la Educación Primaria: Desarrollar hábitos de trabajo individual y de equipo, de esfuerzo y de responsabilidad en el estudio, así como actitudes de confianza en sí mismo, sentido crítico, iniciativa personal, curiosidad, interés y creatividad en el aprendizaje, y espíritu emprendedor; utilizar diferentes representaciones y expresiones artísticas e iniciarse en la construcción de propuestas visuales y audiovisuales; desarrollar sus capacidades afectivas en todos lo ámbitos de la personalidad y en sus relaciones con los demás, así como una actitud contraria a la violencia, a los prejuicios de cualquier tipo y a los estereotipos sexistas. Está vinculada asimismo a los contenidos de las siguientes áreas: Educación Artística, educación Física y Valores Sociales y Cívicos.ES

Stabilization of ω-transaminase from Pseudomonas fluorescens by immobilization techniques

Transaminases are a class of enzymes with promising applications for the preparation and resolution of a vast diversity of valued amines. Their poor operational stability has fueled many investigations on its stabilization due to their biotechnological relevance. In this work, we screened the stabilization of the tetrameric ω-transaminase from Pseudomonas fluorescens (PfωTA) through both carrier-bound and carrier-free immobilization techniques. The best heterogeneous biocatalyst was the PfωTA immobilized as cross-linked enzyme aggregates (PfωTA-CLEA) which resulted after studying different parameters as the precipitant, additives and glutaraldehyde concentrations. The best conditions for maximum recovered activity (29 %) and maximum thermostability at 60 ºC and 70 ºC (100 % and 71 % residual activity after 1 h, respectively) were achieved by enzyme precipitation with 90% acetone or ethanol, in presence of BSA (100 mg/mL) and employing glutaraldehyde (100 mM) as cross-linker. Studies on different conditions for PfωTA-CLEA preparation yielded a biocatalyst that exhibited 31 and 4.6 times enhanced thermal stability at 60 °C and 70 °C, respectively, compared to its soluble counterpart. The PfωTA-CLEA was successfully used in the bioamination of 4-hydroxybenzaldehyde to 4-hydroxybenzylamine. To the best of our knowledge, this is the first report describing a transaminase cross-linked enzyme aggregates as immobilization strategy to generate a biocatalyst with outstanding thermostability.S. Velasco grateful CONACyT for the granted postdoctoral fellowship. L. Betancor, E. Jackson and M. Ripoll acknowledge PEDECIBA, National Research and Innovation Agency of Uruguay (ANII) FSE_1_2016_1_132115 and POS_NAC_2019_1_158182) and Universidad ORT Uruguay. Fernando López acknowledges the funding of IKERBASQUE and Spanish Government (BIO2015-69887-R).Peer reviewe