La casa de piedra en la cuenca del río Alberche

En número dedicado a: La provincia de Ávil

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous

© 2015, Springer Science+Business Media New York. Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9–1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.This work was supported by a Grant from Fondecyt (11060157), and a CONICYT Scholarships to Oriana Flores.Peer Reviewe

Statistical analysis of solid lipid nanoparticles produced by high-pressure homogenization : a practical prediction approach

Lipid nanoparticles (LNPs) are a promising carrier for all administration routes due to their safety, small size, and high loading of lipophilic compounds. Among the LNP production techniques, the easy scale-up, lack of organic solvents, and short production times of the high-pressure homogenization technique (HPH) make this method stand out. In this study, a statistical analysis was applied to the production of LNP by HPH. Spherical LNPs with mean size ranging from 65 nm to 11.623 lm, negative zeta potential under –30 mV, and smooth surface were produced. Manageable equations based on commonly used parameters in the pharmaceutical field were obtained. The lipid to emulsifier ratio (RL/S) was proved to statistically explain the influence of oil phase and surfactant concentration on final nanoparticles size. Besides, the homogenization pressure was found to ultimately determine LNP size for a given RL/S, while the number of passes applied mainly determined polydispersion. a-Tocopherol was used as a model drug to illustrate release properties of LNP as a function of particle size, which was optimized by the regression models. This study is intended as a first step to optimize production conditions prior to LNP production at both laboratory and industrial scale from an eminently practical approach, based on parameters extensively used in formulation

Receptor-targeted nanoparticles modulate cannabinoid anticancer activity through delayed cell internalization

Δ9-tetrahydrocannabinol (Δ9-THC) is known for its antitumor activity and palliative effects. However, its unfavorable physicochemical and biopharmaceutical properties, including low bioavailability, psychotropic side effects and resistance mechanisms associated to dosing make mandatory the development of successful drug delivery systems. In this work, transferring (Tf) surface-modified Δ9-THC-loaded poly(lactide-co-glycolic) nanoparticles (Tf-THC-PLGA NPs) were proposed and evaluated as novel THC-based anticancer therapy. Furthermore, in order to assess the interaction of both the nanocarrier and the loaded drug with cancer cells, a double-fluorescent strategy was applied, including the chemical conjugation of a dye to the nanoparticle polymer along with the encapsulation of either a lipophilic or a hydrophilic dye. Tf-THC PLGA NPs exerted a cell viability decreased down to 17% vs. 88% of plain nanoparticles, while their internalization was significantly slower than plain nanoparticles. Uptake studies in the presence of inhibitors indicated that the nanoparticles were internalized through cholesterol-associated and clathrin-mediated mechanisms. Overall, Tf-modification of PLGA NPs showed to be a highly promising approach for Δ9-THC-based antitumor therapies, potentially maximizing the amount of drug released in a sustained manner at the surface of cells bearing cannabinoid receptors.España Junta Andalucía Project Nr. P09- CTS502

The β-fructofuranosidase from Rhodotorula dairenensis: Molecular cloning, heterologous expression, and evaluation of its transferase activity

The β-fructofuranosidase from the yeast Rhodotorula dairenensis (RdINV) produces a mixture of potential prebiotic fructooligosaccharides (FOS) of the levan-, inulin-and neo-FOS series by transfructosylation of sucrose. In this work, the gene responsible for this activity was characterized and its functionality proved in Pichia pastoris. The amino acid sequence of the new protein contained most of the characteristic elements of β-fructofuranosidases included in the family 32 of the glycosyl hydrolases (GH32). The heterologous yeast produced a protein of about 170 kDa, where N-linked and O-linked carbohydrates constituted about 15% and 38% of the total protein mass, respectively. Biochemical and kinetic properties of the heterologous protein were similar to the native enzyme, including its ability to produce prebiotic sugars. The maximum concentration of FOS obtained was 82.2 g/L, of which 6-kestose represented about 59% (w/w) of the total products synthesized. The potential of RdINV to fructosylate 19 hydroxylated compounds was also explored, of which eight sugars and four alditols were modified. The flexibility to recognize diverse fructosyl acceptors makes this protein valuable to produce novel glycosyl-compounds with potential applications in food and pharmaceutical industriesThis work was supported by Spanish Ministry of Economy and Competitiveness: BIO2016- 76601-C3-2 and of Science and Innovation PID2019-105838RB-C32; Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences); and by institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. CBMSO Proteomics Facility belongs to ProteoRed PRB2-ISCIII and was supported by the grant PT13/0001. M.G.-P. was supported by the Spanish Ministry of Education’s University Personnel Training Plan (FPU

Interés terapéutico de cannabinoides: análisis bibliométrico en Pubmed, Scopus y web of Science

En este trabajo se ha analizado la actividad científica mundial sobre la aplicación terapéutica de cannabinoides en diferentes patologías, así como la posible aplicación de la nanotecnología en el desarrollo de nuevos sistemas de administración para este tipo de fármacos. Para ello, se han utilizado tres bases de datos habituales en Ciencias de la Salud: PubMed, Scopus y Web of Science (WoS). Los resultados del análisis muestran claramente un interés creciente en estas moléculas y sus posibles aplicaciones terapéuticas. El análisis se ha hecho comparando los resultados para el ítem cannabinoids en las tres bases de datos y su asociación con therapeutic. Como se muestra en el trabajo, para Scopus y WoS se obtienen tendencias similares aunque el número de documentos recogidos en Scopus entre 2007-2012 es menor que en WOS. Por el contrario, PubMed muestra una tendencia totalmente discordante. Respecto a la aplicación de la nanotecnología en el desarrollo de nuevos sistemas de administración de estos fármacos, Scopus no diferencia los resultados para los ítems nanotechnology y microspheres. Para la asociación nanoparticles + cannabinoids, Scopus y WoS es la que vuelca más resultados, con un total de 10 artículos de investigación, siendo el 40 % de ellos españole

Antagonistic Toxic Effects of Surfactants Mixtures to Bacteria Pseudomonas putida and Marine Microalgae Phaeodactylum tricornutum

Surfactants can be found in an ever-widening variety of products and applications, in which the combination of several types of surfactants is used to reinforce their properties, looking for synergistic effects between them. After use, they tend to be discarded into wastewater, ending up in aquatic bodies with concerning harmful and toxic effects. The aim of this study is the toxicological assessment of three anionic surfactants (ether carboxylic derivative, EC) and three amphoteric surfactants (amine-oxide-based, AO), individually and in binary mixtures of them (1:1 w/w), to bacteria Pseudomonas putida and marine microalgae Phaeodactylum tricornutum. Critical Micelle Concentration (CMC) was determined to demonstrate the capacity to reduce surface tension and the toxicity of the surfactants and mixtures. Zeta potential (ζ-potential) and micelle diameter (MD) were also determined to confirm the formation of mixed surfactant micelles. The Model of Toxic Units (MTUs) was used to quantify the interactions of surfactants in binary mixtures and to predict if the concentration addition or response addition principle can be assumed for each mixture. The results showed a higher sensitivity of microalgae P. tricornutum to the surfactants tested and their mixtures than bacteria P. putida. Antagonism toxic effects have been detected in the mixture of EC + AO and in one binary mixture of different AOs; this is to say, the mixtures showed lower toxicity than expected.Ministry of Universities of the Spanish Government within the predoctoral grant FPU (Ayudas para la Formacion de Profesorado Universitario) FPU17/0335

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos. Procedimiento de obtención de un biocatalizador, que comprende: inmovilizar una enzima fúngica por inclusión en un gel de alginato cálcico; y el secado posterior el biocatalizador inmovilizado obtenido en el paso (a). La invención también se refiere al biocatalizador obtenido por el procedimiento de la invención y que comprende enzimas fúngicas, preferiblemente fructosiltransferasa o - fructofuranosidasa, inmovilizadas en alginato. Además la invención se refiere al uso de dicho biocatalizador para la biotransformación en las que el sustrato es una disolución concentrada de un carbohidrato y se puede llevar a cabo en un reactor continuo.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridA1 Solicitud de patente con informe sobre el estado de la técnic

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous ¿-glucosidase with potential for prebiotics production

Abstract Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α–(1–6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583–602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-lucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme’s activity against long-size substrates such as maltoheptaose and soluble starch.Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biología Molecular Severo OchoaPeer Reviewe

Regioselective synthesis of neo-erlose by the beta-fructofuranosidase from Xanthophyllomyces dendrorhous

The beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the 6G-series), which contain a beta(2-6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6’-O--fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.Projects BIO2010-20508-C04-01 and BIO2010-20508-C04-04 from Spanish Ministry of Science and Innovation supported this research. We thank Fundación Ramon Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa.Peer reviewe