Controlled manipulation of enzyme specificity through immobilization-induced flexibility constraints

It is thought that during immobilization enzymes, as dynamic biomolecules, may become distorted and this may alter their catalytic properties. However, the effects of different immobilization strategies on enzyme rigidity or flexibility and their consequences in specificity and stereochemistry at large scale has not been yet clearly evaluated and understood. This was here investigated by using as model an ester hydrolase, isolated from a bacterium inhabiting a karstic lake, with broad substrate spectrum (72 esters being converted; 61.5 U mg$^{-1}$ for glyceryl tripropionate) but initially non-enantiospecific. We found that the enzyme (7 nm × 4.4 nm × 4.2 nm) could be efficiently ionic exchanged inside the pores (9.3 nm under dry conditions) of amino-functionalized ordered mesoporous material (NH$_{2}$-SBA-15), achieving a protein load of 48 mg g−1, and a specific activity of 4.5 ± 0.1 U mg$^{-1}$. When the enzyme was site-directed immobilized through His interaction with an immobilized cationon the surface of two types of magnetic micro-particles through hexahistidine-tags, protein loads up to 10.2 μg g$^{-1}$ and specific activities of up to 29.9 ± 0.3 U mg$^{-1}$, were obtained. We found that ionically exchanged enzyme inside pores of NH2-SBA-15 drastically narrowed the substrate range (17 esters), to an extent much higher than ionically exchanged enzyme on the surface of magnetic micro-particles (up to 61 esters). This is attributed to differences in surface chemistry, particle size, and substrate accessibility to the active site tunnel. Our results also suggested, for the first time, that immobilization of enzymes in pores of similar size may alter the enzyme structures and produce enzyme active centers with different configuration which promote stereochemical conversions in a manner different to those arising from surface immobilization, where the strength of the ionic exchange also has an influence. This was shown by demonstrating that when the enzyme was introduced inside pores with a diameter (under dry conditions) slightly higher than that of the enzyme crystal structure a biocatalyst enantiospecific for ethyl (R)-4-chloro-3-hydroxybutyrate was produced, a feature not found when using wider pores. By contrast, immobilization on the surface of ferromagnetic microparticles produced selective biocatalysts for methyl (S)-(+)-mandelate or methyl (S)-lactate depending on the functionalization. This study illustrates the benefits of extensive analysis of the substrate spectra to better understand the effects of different immobilization strategies on enzyme flexibility/rigidity, as well as substrate specificity and stereochemistry. Our results will help to design tunable materials and interfaces for a controlled manipulation of specificity and to transform non-enantiospecific enzymes into stereo-chemically substrate promiscuous biocatalysts capable of converting multiple chiral molecules

Infrared Fluorescent Imaging as a Potent Tool for In Vitro, Ex Vivo and In Vivo Models of Visceral Leishmaniasis

Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic diseaseThis research was supported by Ministerio de Economía y Competitividad (www.mineco.gob.es) grants AGL2010-16078/GAN to RBF and CYTED 214RT0482 to RMR; Instituto de Salud Carlos III (www.isciii.es) grants PI12/00104 to RMR and RICET RD12/0018/0004 to MF; Junta de Castilla y León (www.jcyl.es) grants Gr238 and LE182U13; European Commision (cordis.europa.eu/home_es.html), grant HOMIN - 317057-FP7-PEOPLE-2012-ITN and BIOIMID (http://www.fundacionareces.es) Proyecto de Excelencia Instituto Sanitario “La Princesa” and Fundación Ramón Areces to MF. SK is granted from AECC Foundation (https://www.aecc.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the anuscrip

A Sensitivity Analysis for Quality Measures of Quantitative Association Rules

There exist several fitness function proposals based on a combination of weighted objectives to optimize the discovery of association rules. Nevertheless, some differences in the measures used to assess the quality of association rules could be obtained according to the values of such weights. Therefore, in such proposals it is very important the user’s decision in order to specify the weights or coefficients of the optimized objectives. Thus, this work presents an analysis on the sensitivity of several quality measures when the weights included in the fitness function of the existing QARGA algorithm are modified. Finally, a comparative analysis of the results obtained according to the weights setup is provided.MICYT TIN2011-28956-C02-00Junta de Andalucía P11-TIC-752

Efectos del vibrado del pino piñonero (Pinus pinea L.) en el vigor de los árboles: densidad de copa, crecimiento de guías y parásitos de debilidad

In the Northwest of Spain the mechanized harvesting of pine cones is more and more frequenIn order to quantify the effects of vibration on the vigor of the trees, parcels of stone pine were planted in plateau and countryside. Both the mechanized and manual harvests were analyzed in adult and young trees. The growth of the tree shoots was measured. The presence and abundance of three insect plagues was analyzed in those same trees: Tomicus piniperda, Rhyacionia buoliana and Thaumetopoea pityocampa. The average shoot length of those trees manually harvested was superior to that of those mechanically harvested. The difference was very significant in young trees (40-50 years old) in the countryside and in adult trees (more than 80 years) on the plateau. Rhyacionia buoliana was more abundant in adult trees that young trees, but the relation of its abundance with the harvest method was not seen. The presence of Tomicus piniperda was rare and was only detected in mechanically harvested stands. Thaumetopoea pityocampa was more frequent in trees harvested by hand than those harvested mechanically and was more abundant in young stands. The percentage tree of defoliation was low in all stands independent of harvest method.En el noroeste de España es cada vez más frecuente la recolección mecanizada de piña. Para cuantificar los efectos del vibrado sobre el vigor de los árboles se instalaron en montes de páramo y de campiña parcelas donde se realizó cosecha mecanizada y manual en arbolado adulto y joven. Se midió el crecimiento de los brotes y se analizó la presencia y abundancia de tres insectos plaga sobre estos árboles, Tomicus piniperda y Rhyacionia buoliana, considerados parásitos de debilidad, así como Thaumetopoea pityocampa. La longitud media de los brotes fue superior en árboles cosechados manualmente. La diferencia es importante en árboles jóvenes (40-50 años) de campiñas y en árboles adultos (más de 80 años) de páramos. Rhyacionia buoliana fue más abundante en árboles adultos que en arbolado joven, pero no se vio relación de su abundancia con el método de cosecha. Tomicus piniperda fue escaso y sólo se detectó en rodales cosechados mecanizadamente. Thaumetopoea pityocampa es más frecuente en rodales cosechados manualmente, y en rodales jóvenes lo que indica que selecciona los árboles de crecimiento más vigoroso para alimentarse. Los porcentajes de defoliación fueron bajos en todas las parcelas

Visions of the World by Pepper and its application to Health Education

INTRODUCCIÓN: Las metáforas impregnan nuestra vida cotidiana y no sólo el lenguaje también el pensamiento y la acción. La enfermera usa las metáforas como recurso didáctico para facilitar la comunicación y la comprensión de conceptos abstractos. Los usuarios expresan sus vivencias de salud o enfermedad a través de metáforas de su ámbito personal y cotidiano. La comprensión del uso de las metáforas en Educación para la Salud (EpS) requiere examinar también los paradigmas dentro de los cuales las metáforas son construidas. Este estudio pretende conocer las metáforas básicas o “visones del mundo” que utilizan la enfermera en la consulta de atención primaria en sus intervenciones educativas-asistenciales con usuarios afectados enfermedades crónicas

Active Q-switched Fiber Lasers with Single and Dualwavelength Operation

A brief explanation on Q-switched fiber laser operating principle for active technique in terms of operation characteristics is presented. Experimental analysis of proposed pulsed fiber lasers by the active Q-switched technique is demonstrated. Experimental setups include the use of Er/Yb doped fiber as a gain medium and an acousto-optic modulator as cavity elements. Setup variations include the use of fiber Bragg gratings for wavelength selection and tuning and Sagnac interferometer for wavelength selection in single wavelength operation and for cavity loss adjustment in dual wavelength operation. The experimental analysis of principal characteristics of single-wavelength operation of the fiber laser and cavity loss adjustment method for dual-wavelength laser operation are discussed

B-Function Expression in the Flower Center Underlies the Homeotic Phenotype of Lacandonia schismatica (Triuridaceae)

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa

Sparse deconvolution using support vector machines

Sparse deconvolution is a classical subject in digital signal processing, having many practical applications. Support vector machine (SVM) algorithms show a series of characteristics, such as sparse solutions and implicit regularization, which make them attractive for solving sparse deconvolution problems. Here, a sparse deconvolution algorithm based on the SVM framework for signal processing is presented and analyzed, including comparative evaluations of its performance from the points of view of estimation and detection capabilities, and of robustness with respect to non-Gaussian additive noise.Publicad

Hospitalization budget impact during the COVID-19 pandemic in Spain

To Mrs. Anne Murray for her support to translate the manuscript. This article is part of the doctoral thesis of Laura Álvarez as part of the Doctoral Program in Pharmacy, Granada University (Spain).Objectives: The aim was to determine the direct impact of the COVID-19 pandemic on Spain’s health budget. Methods: Budget impact analyses based on retrospective data from patients with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) admitted to a Spanish hospital between February 26 and May 21, 2020. Direct medical costs from the perspective of the hospital were calculated. We analyzed diagnostic tests, drugs, medical and nursing care, and isolation ward and ICU stays for three cohorts: patients seen in the emergency room only, hospitalized patients who tested positive for SARS-CoV-2, and patients who tested negative. Results: The impact on the hospital’s budget for the 3 months was calculated at €15,633,180, 97.4% of which was related to health care and hospitalization. ICU stays accounted for 5.3% of the total costs. The mean cost per patient was €10,744. The main costs were staffing costs (10,131 to 11,357 €/patient for physicians and 10,274 to 11,215 €/patient for nurses). Scenario analysis showed that the range of hospital expenditure was between €14,693,256 and €16,524,924. The median impact of the pandemic on the Spanish health budget in the sensitivity analysis using bootstrapped individual data was €9357 million (interquartile range [IQR], 9071 to 9689) for the conservative scenario (113,588 hospital admissions and 11,664 ICU admissions) and €10,385 million (IQR, 110,030 to 10,758) for the worst-case scenario (including suspected cases). Conclusion: The impact of COVID-19 on the Spanish public health budget (12.3% of total public health expenditure) is greater than multiple sclerosis, cancer and diabetes cost