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Las nuevas tecnologías de la información han transformado la manera de prestar la asistencia en los servicios de salud, impregnando casi todos los aspectos de la atención sanitaria. A medida que la complejidad del sistema aumenta, es más difícil trabajar de manera óptima sin la asistencia de las nuevas tecnologías. Su implantación supone un avance, bien por el adelanto que entraña el uso adecuado de cualquier nueva tecnología en el cuidado de la salud, bien por el desarrollo de aplicaciones específicas que mejoran la seguridad de la asistencia. Sin embargo, factores como un diseño incorrecto, implementación y mantenimiento deficientes, capacitación inadecuada, junto al exceso de confianza y dependencia, pueden hacer que las tecnologías comprometan, más que favorecer, la seguridad del paciente. Este artículo describe los efectos beneficiosos, y los que no lo son tanto, de la introducción en nuestro país de la historia clínica y la receta electrónicas en la calidad y la seguridad de la asistencia sanitaria.

The electronic prescription has meant an evolution in the quality of health care, but it has a series of deficiencies that compromise the adherence to treatments. The objective of this work was to collect and group these limitations and propose the changes that could solve them.During 2017 and 2018, through dispensing and pharmacotherapeutic monitoring activities, incidents related to the electronic prescription were collected in a pharmacy in Santander and their causes were identified. A survey was conducted and sent to the Information Centers of the Medicines of the rest of the regions to know if the same problems were shared and the resulting data were treated by frequency distribution.We detected 1,500 incidents related to the way of access to the treatment and its activation period, with the availability of the medication when the patient requested it and those in which the assessment of the problem by the doctor was essential. The survey data showed that in 100% of the CCAA the patient must return to the doctor when there is an incidence, because there is no protocol to solve it from the pharmacy.These incidents compromise the adherence to treatments and the health of patients. To avoid them, we propose the elimination of the password that is necessary to access the treatment, the establishment of a minimum period of 30 days for its activation, the creation of protocols so that the pharmacist can solve problems and perform dispensations in special circumstances, and finally, the optimization of the communication between pharmacist and doctor.La receta electrónica ha supuesto una evolución en la calidad de la asistencia sanitaria, pero presenta una serie de deficiencias que comprometen la adherencia a los tratamientos. El objetivo de este trabajo fue recopilar y agrupar estas limitaciones y proponer cambios que podrían resolverlas.Durante los años 2017 y 2018, a través de las actividades de dispensación y seguimiento farmacoterapéutico, se recogieron en una farmacia de Santander las incidencias relacionadas con la dispensación de recetas electrónicas y se identificaron sus causas. Se realizó una encuesta que se envió a los Centros de Información de los Medicamentos del resto de comunidades autónomas para conocer si se compartían los mismos problemas y se trataron los datos resultantes mediante distribución de frecuencias.Se detectaron 1.500 incidencias relacionadas con el modo de acceso al tratamiento y su periodo de activación, con la disponibilidad de la medicación cuando el paciente lo solicitaba y aquellas en las que era indispensable la valoración del problema por parte del médico. Los datos de la encuesta mostraron que en el 100% de las comunidades autónomas el paciente debía volver al centro de salud para solucionar estas incidencias, al no existir ningún protocolo para resolverlas desde la farmacia.Estas incidencias comprometen la adherencia a los tratamientos y la salud de los pacientes. Para evitarlas, proponemos la eliminación del número de orden que es necesario para acceder al tratamiento, la instauración de un período mínimo de 30 días para su activación, la creación de protocolos para que el farmacéutico pueda solucionar problemas y realizar dispensaciones en circunstancias especiales, y por último, la optimización de la comunicación entre farmacéutico y médico.

Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10,000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.

Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).

This is an Open Access article distributed under the terms of the Creative Commons Attribution License., Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC-DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in anic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxasemediated integration in any target genome., Spanish Ministry of Science and Innovation [BIO2010-14809 to G.M., BFU2011-26608 to F.C.]; European Union Seventh Framework Programme [612146/FP7-ICT-2013, 282004/FP7-HEALTH.2011.2.3.1-2 to F.C.]. Funding for open access charge: Spanish Ministry of Science and Innovation Grants [BIO2010-14809] and [BFU2011-26608].

The Morita–Baylis–Hillman reaction of cyclohexenone and p -nitro benzaldehyde is catalyzed by carrier proteins such as serum albumins or enzymes such as certain lipases, conversion of up to 35% and enantioselectivities of up to 19% being observed.

TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA-TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their conjugation efficiency. © 2009 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.

Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexyl-2-methylbuta-2,3-dienoate (E = 111); the high enantioselectivity of the Leu162Phe mutant was rationalized by π–π stacking.

In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.

Mutants of the lipase from Bacillus subtilis, previously engineered for enhanced thermostability using directed evolution based on the B-FIT method, show significantly increased tolerance to hostile organic solvents.