Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation

“This is the pre-peer reviewed version of the following article: Iturrate, L., Sánchez-Moreno, I., Oroz-Guinea, I., Pérez-Gil, J., García-Junceda, E. (2010) “Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation“ Chem. Eur. J., 16, 4018-4030, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/chem.200903096/abstract”A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channellingWe thank the Spanish Ministerio de Ciencia e Innovación for financial support (Grant CTQ2007-67403/BQU). J. P.-G. has been supported by grants BIO2009-09694 and CSD2007-00010. L. Iturrate and I. Sánchez-Moreno acknowledges the Predoctoral Fellowship from Comunidad de Madrid. I. Oroz-Guinea is a JAEPredoc fellow from CSIC. We thank E. G. Doyagüez for her assistance on NMR characterization

EEG Single-Trial Classification of Visual, Auditive and Vibratory Feedback Potentials in Brain-Computer Interfaces

Feedback stimuli are fundamental components in Brain-Computer Interfaces. It is known that the presentation of feedback stimuli elicits certain brain potentials that can be measured and classified. As stimuli can be given through different sensory modalities, it is important to understand the effects of different types of feedback on brain responses and their impact on classification. This paper presents a protocol used to obtain brain potentials elicited by visual, auditive or vibrotactile feedback stimuli. Experiments were carried out with five different subjects for each modality. Four different single-trial classification strategies were compared, according to the information used to train the classifier, achieving a classification rate of approximately 80% for each modality

Single-Trial Classification of Feedback Potentials within Neurofeedback Training with an EEG Brain-Computer Interface

Neurofeedback therapies are an emerging technique used to treat neuropsychological disorders and to enhance cognitive performance. The feedback stimuli presented during the therapy are a key factor, serving as guidance throughout the entire learning process of the brain rhythms. Online decoding of these stimuli could be of great value to measure the compliance and adherence of the subject to the training. This paper describes the modeling and classification of performance feedback potentials with a Brain-Computer Interface (BCI), under a real neurofeedback training with five subjects. LDA and SVM classification techniques are compared and are both able to provide an average performance of approximately 80%

Quantitative assessment of energy and resource recovery in wastewater treatment plants based on plant-wide simulations.

The growing development of technologies and processes for resource treatment and recovery is offering endless possibilities for creating new plant-wide configurations or modifying existing ones. However, the configurations’ complexity, the interrelation between technologies and the influent characteristics turn decision-making into a complex or unobvious process. In this frame, the Plant-Wide Modelling (PWM) library presented in this paper allows a thorough, comprehensive and refined analysis of different plant configurations that are basic aspects in decision-making from an energy and resource recovery perspective. In order to demonstrate the potential of the library and the need to run simulation analyses, this paper carries out a comparative analysis of WWTPs, from a techno-economic point of view. The selected layouts were (1) a conventional WWTP based on a modified version of the Benchmark Simulation Model No. 2, (2) an upgraded or retrofitted WWTP, and (3) a new Wastewater Resource Recovery Facilities (WRRF) concept denominated as C/N/P decoupling WWTP. The study was based on a preliminary analysis of the organic matter and nutrient energy use and recovery options, a comprehensive mass and energy flux distribution analysis in each configuration in order to compare and identify areas for improvement, and a cost analysis of each plant for different influent COD/TN/TP ratios. Analysing the plants from a standpoint of resources and energy utilization, a low utilization of the energy content of the components could be observed in all configurations. In the conventional plant, the COD used to produce biogas was around 29%, the upgraded plant was around 36%, and 34% in the C/N/P decoupling WWTP. With regard to the self-sufficiency of plants, achieving self-sufficiency was not possible in the conventional plant, in the upgraded plant it depended on the influent C/N ratio, and in the C/N/P decoupling WWTP layout self-sufficiency was feasible for almost all influents, especially at high COD concentrations. The plant layouts proposed in this paper are just a sample of the possibilities offered by current technologies. Even so, the library presented here is generic and can be used to construct any other plant layout, provided that a model is available

Leveraging clinical decision support tools to improve guideline-directed medical therapy in patients with atherosclerotic cardiovascular disease at hospital discharge

Background: Guidelines recommend moderate to high-intensity statins and antithrombotic agents in patients with atherosclerotic cardiovascular disease (ASCVD). However, guideline-directed medical therapy (GDMT) remains suboptimal. Methods: In this quality initiative, best practice alerts (BPA) in the electronic health record (EHR) were utilized to alert providers to prescribe to GDMT upon hospital discharge in ASCVD patients. Rates of GDMT were compared for 5 months pre- and post-BPA implementation. Multivariable regression was used to identify predictors of GDMT. Results: In 5985 pre- and 5568 post-BPA patients, the average age was 69.1 ± 12.8 years and 58.5% were male. There was a 4.0% increase in statin use from 67.3% to 71.3% and a 3.1% increase in antithrombotic use from 75.3% to 78.4% in the post-BPA cohort.  Conclusions: This simple EHR-based initiative was associated with a modest increase in ASCVD patients being discharged on GDMT. Leveraging clinical decision support tools provides an opportunity to influence provider behavior and improve care for ASCVD patients, and warrants further investigation

The first 20 months of the COVID-19 pandemic: Mortality, intubation and ICU rates among 104,590 patients hospitalized at 21 United States health systems

Main objective There is limited information on how patient outcomes have changed during the COVID-19 pandemic. This study characterizes changes in mortality, intubation, and ICU admission rates during the first 20 months of the pandemic. Study design and methods University of Wisconsin researchers collected and harmonized electronic health record data from 1.1 million COVID-19 patients across 21 United States health systems from February 2020 through September 2021. The analysis comprised data from 104,590 adult hospitalized COVID-19 patients. Inclusion criteria for the analysis were: (1) age 18 years or older; (2) COVID-19 ICD-10 diagnosis during hospitalization and/or a positive COVID-19 PCR test in a 14-day window (+/- 7 days of hospital admission); and (3) health system contact prior to COVID-19 hospitalization. Outcomes assessed were: (1) mortality (primary), (2) endotracheal intubation, and (3) ICU admission. Results and significance The 104,590 hospitalized participants had a mean age of 61.7 years and were 50.4% female, 24% Black, and 56.8% White. Overall risk-standardized mortality (adjusted for age, sex, race, ethnicity, body mass index, insurance status and medical comorbidities) declined from 16% of hospitalized COVID-19 patients (95% CI: 16% to 17%) early in the pandemic (February-April 2020) to 9% (CI: 9% to 10%) later (July-September 2021). Among subpopulations, males (vs. females), those on Medicare (vs. those on commercial insurance), the severely obese (vs. normal weight), and those aged 60 and older (vs. younger individuals) had especially high mortality rates both early and late in the pandemic. ICU admission and intubation rates also declined across these 20 months. Conclusions Mortality, intubation, and ICU admission rates improved markedly over the first 20 months of the pandemic among adult hospitalized COVID-19 patients although gains varied by subpopulation. These data provide important information on the course of COVID-19 and identify hospitalized patient groups at heightened risk for negative outcomes. Trial registration ClinicalTrials.gov Identifier: NCT04506528 (https://clinicaltrials.gov/ct2/show/NCT04506528)

Nueva Metodología de Modelado Matemático Integral de las EDAR

Tras una revisión del potencial y las limitaciones de los principales modelos matemáticos existentes para describir de manera conjunta los procesos unitarios de las Estaciones Depuradoras de Aguas Residuales (EDAR), la presente tesis desarrolla una nueva metodología de modelado. Esta metodología permite la construcción sistemática y rigurosa de modelos integrales de planta capaces de reproducir las mutuas interrelaciones entre los diferentes elementos de las líneas de aguas y fangos, garantizando en todo momento la continuidad de masa elemental y carga. La nueva metodología desarrollada, denominada metodología PWM (Plant Wide Modelling) está basada en la utilización de una lista genérica de transformaciones de las que se eligen las más adecuadas para los procesos que tienen lugar en cada EDAR objeto de estudio. La definición de los componentes en sus fracciones másicas elementales permite garantizar la continuidad de masa y carga en cada transformación y a lo largo de toda la planta. La definición de los componentes en base a sus fracciones másicas ha sido utilizada para el desarrollo de un algoritmo para la caracterización automática del influente. Este algoritmo ha sido empleado con éxito en la caracterización del agua residual influente de la EDAR de Galindo (Bilbao). La metodología PWM propuesta ha sido utilizada para la construcción de un modelo integral para un caso de estudio estándar (BSM2). El modelo obtenido ha sido verificado en diferentes escenarios de simulación. Posteriormente se ha llevado a cabo un análisis comparativo entre la utilización del modelo de EDAR obtenido frente a otras propuestas de modelado integral existentes en la bibliografía. Se ha construido en modelo integral para la EDAR Galindo-Bilbao de acuerdo con la metodología PWM. El modelo obtenido ha sido utilizado en la validación de una estrategia de control automático en la línea de aguas de la EDAR. Posteriormente se ha llevado a cabo una exploración de la operación de la EDAR en escenarios hipotéticos que consideran de forma conjunta líneas de aguas y fangos. La exposición finaliza con el capítulo dedicado a las conclusiones del estudio realizado, la bibliografía utilizada y una serie de anexos que proporcionan información adicional a la incluida en la memoria

Proteína quimérica aldolasa/quinasa, procedimiento de obtención y sus aplicaciones

Proteína quimérica aldolasa/quinasa, procedimiento de obtención y sus aplicaciones. La presente invención describe una nueva proteína quimérica que reúne en una única cadena polipeptídica dos actividades enzimáticas: una aldolasa y una quinasa, útil en la formación de enlaces C-C entre una cetona donadora y un aldehído aceptor complementándose el sistema con la regeneración in situ del ATP. Así, esta proteína quimérica permite desarrollar nuevos compuestos químicos con actividad biológica, por ejemplo, carbohidratos y análogos.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patentes con informe sobre el estado de la técnic

DHAP-dependent aldolases in the core of multistep processes

Capítulo 4, pp 61-81The aldol addition reaction has long been recognized as one of the most useful tools that has the synthetic chemist for the construction of new carbon-carbon bonds. The enzyme aldolases are among the most important biocatalysts for C–C bond formations in nature. Dihydroxyacetone phosphate-dependent aldolases have been extensively used in chemoenzymatic syntheses of carbohydrate, carbohydrate-like structures, or non-carbohydrate compounds. This review will be mainly focused on that applications in which DHAP-dependent aldolase are part of a multi-enzyme system, or alternatively on those in which the aldolase catalysed reaction is key in a multi-step synthetic pathway.Peer reviewe

New heat transfer and operating cost models for the Plant-Wide simulation of full-scale WWTPs.

Tesi honen helburu nagusia, modelo matematikoak eraikitzeko beharrezkoa den prozedura zorrotz eta sistematikoa garatu ata balioztatzea izan da, hondakin uren araztegia (HUA) osatzen duten prozesu ororen bero transferentzia eta eragiketa-kostuak deskribatu ahal izateko. Helburu hau erdiesteko asmoz, tesiak modelaketa metodologia berri bat aurkezten du, unitateetan eman daitezkeen erreakzio biokimiko, kimiko eta fisiko-kimikoek sor ditzaketen bero aldaketak zenbatezteko. Metodologia, fase (likido, gas nahiz solido) bakoitzeko erreakzio-beroen zenbatezpenean oinarritzen da, formazio entalpiak erabiliz eta Hess-en legea aplikatuz. Plant-Wide Modelling (PWM) metodologiak eskaintzen duen osagaien karakterizazio xehatuak, osagai bakoitzaren formazio entalpiaren zenbatespena ahalbidetzen du, baita erreakzio-beroaren kalkulu sistemiko eta dinamikoa ere, uneoro planta osoan zehar energia termikoaren ata masikoaren jarraitasuna bermatuz. Metodologia hau, bero transferentzia modelo orokor batetara gehitu da prozesuko unitateetan aurki daitezkeen faseen temperaturak iragartzeko asmoz. Eragiketa-kostu modeloei dagokienez, tesiak ingeniaritzan erabiltzen diren adierazpenetan oinarritutako eragingailuen bilduma zabala aurkezten du. Adierazpen hauen erabilerak, eragiketa-kostuen zenbatezpen zehatz eta errealista ahalbidetzen du, hauek prozesuko aldagaiekin (emari, erreakzio entalpia, solidoen kontzentrazio, etab.) duten lotura estuari esker. Tesi honetan garatutako bero transferentzia eta kostu modeluek, eta Lizarralde eta kol-ek (2015) aurkeztutako modelu fisiko-kimikoak, Ceit-IK4 PWM metodologia eguneratzen lagundu dute, Extended Plant-Wide Modelling (E-PWM) metodología izenekora. Erreakzio-beroaren zenbatezpenerako garatutako modelaketa metodologia, datu teoriko eta experimentalekin balioztatu da. Bero transferentzia modeloaren iragartze ahalmena berriz, digestio aerobio termofilo eta autoiraunkor (DATA) baten portaeraren simulazio bitartez egiaztatu da. Modelo termiko orokorraren baliagarritasuna eta aplikagarritasuna frogatzeko, simulazio bidezko bi azterketa-kasu burutu dira, lehenengoan DATA erreaktorea simulatuz eta bigarrengoan erreferentzizko planta global bat erabiliz (BSM2). Modelo bildumaren ahalmena erakusteko asmoz, hiru HUA ebolutibo alderatu dira ikuspuntu tekniko-ekonomiko batetatik. Aukeratutako konfigurazioak ondorengoak dira: (1) Ohiko hondakin uren araztegi bat erreferentzizko bigarren simulazio modelo (BSM2) eraldatuan oinarritua, (2) Ohiko HUAren bertsio hobetu eta berhornitua, eta (3) arazketa kontzeptu berri batean oinarritutako konfigurazio bat, C/N/P banandua duen arategia izenekoa. Ondoren, hiru HUA errealetan burututako azterketa-kasuak aurkezten dira modelo bilduma egokitu eta malguen baliagarritasuna erakusteko, hau da, PWM modelo bildumaren baliagarritasuna. Bukatzeko, tesiaren ondorio nagusiak biltzen dituen atala, erabilitako erreferentzi bibliografiko zehatzen zerrenda eta tesian aurkeztutako informazioa osatzen duten zenbait eranskin gehitu dira.El principal objetivo de esta tesis ha sido el de desarrollar y validar un procedimiento riguroso y sistemático para construir modelos matemáticos que describan las transferencias de calor y los costes operacionales en estaciones depuradoras de aguas residuales (EDARs). Con el fin de lograr este objetivo, la tesis presenta una nueva metodología de modelado para el cálculo de las variaciones de calor producidas por las reacciones bioquímicas, químicas y físico-químicas en cualquier unidad. La metodología se basa en la estimación del calor de reacción de cada fase (líquida, sólida o gaseosa) mediante las entalpias de formación de los componentes del modelo y aplicando la Ley de Hess. La detallada caracterización de los componentes que ofrece la metodología Plant-Wide Modelling (PWM), permite la estimación de las entalpías de formación de cada componente del modelo, y hace posible un cálculo sistemático y dinámico del calor liberado o absorbido por cada transformación, garantizando en todo momento la continuidad de energía calorífica en paralelo con la continuidad de masa en cualquier punto de la planta. Esta metodología ha sido incorporada a un completo y genérico modelo de transferencias de calor para la predicción de la temperatura de cualquier fase (líquida, sólida o gaseosa) presente en las unidades de proceso. En lo que respecta a los modelos de costes de operación, la tesis presenta una amplia librería de modelos de actuadores basados en ecuaciones ingenieriles. La utilización de expresiones ingenieriles ha permitido tener una estimación más detallada y realista de los costes operacionales, por el hecho de estar éstos asociados a variables operacionales de proceso (caudales, entalpías de reacción, concentración de sólidos, etc.). Los modelos de transferencias de calor y los modelos de costes operacionales desarrollados en esta tesis, junto con el modelo físico-químico desarrollado por Lizarralde et al., (2015) han servido para actualizar la metodología Ceit-IK4 PWM, a una nueva versión de la metodología denominada Extended Plant-Wide Modelling (E-PWM). La metodología de modelado desarrollada para el cálculo de las variaciones de calor producidas por las transformaciones ha sido validada mediante una comparación con datos experimentales y teóricos bibliográficos. Mientras que la verificación de la capacidad predictiva del modelo de transferencias de calor se ha llevado a cabo mediante la simulación del comportamiento dinámico de un digestor aerobio termófilo auto-sostenido (ATAD). Para testear la utilidad y aplicabilidad del modelo térmico global, se han realizado dos estudios por simulación, uno de ellos en el mismo reactor ATAD y otro en una planta global de referencia (BSM2). Con el objetivo de mostrar el potencial de la librería, se han comparado tres plantas evolutivas desde un punto de vista técnico-económico. Las configuraciones seleccionados han sido: (1) una EDAR convencional basada en una versión modificada del modelo de simulación de referencia No. 2 (BSM2), (2) una versión mejorada o reequipada de la EDAR convencional, y (3) un nuevo concepto de planta denominado EDAR con desacople de C/N/P. Posteriormente, la tesis presenta tres casos de estudio realizados en plantas de tratamiento de aguas residuales a escala real con el objetivo de mostrar la utilidad de las librerías de modelos adaptadas y flexibles frente a optimizaciones de plantas a gran escala, como es el caso de la librería de modelos PWM. La exposición finaliza con un capítulo dedicado a las conclusiones más significativas, la bibliografía utilizada y una serie de anexos que proporcionan información adicional a la incluida en la memoria, como por ejemplo la descripción detallada de los modelos y la caracterización de los componentes del modelo.The main objective of this thesis has been to develop and validate a systematic and rigorous procedure for constructing mathematical models describing both heat transfers and operating costs in Wastewater Treatment Plants (WWTPs). In order to achieve this objective, the thesis presents a new modelling methodology for calculating the heat variations produced by biochemical, chemical and physico-chemical transformations in any unit. The methodology is based on the estimation of the heat of reaction of each phase (liquid, solid or gaseous) by the enthalpies of formation of model components, applying the Hess's law. The detailed characterization of the components that provides the Plant-Wide Modelling (PWM) methodology, enables the estimation of the enthalpy of formation for each model component and makes possible a systematic and dynamic calculation of the heat released or absorbed by each transformation, guaranteeing heat energy continuity in parallel with the mass continuity at any point in the plant. This methodology has been incorporated into a complete and generic heat transfer model for predicting the temperature of any phase present in the unit-processes. Regarding the operating cost models, the thesis presents an extensive library of actuator models based on engineering equations. The engineering expressions adapted depend closely on the operational variables of the process (solids concentration, flowrates, enthalpy changes of reaction, etc.), allowing a more detailed and realistic estimate. The heat transfer and operating costs models developed in this thesis, along with the physico-chemical model developed by Lizarralde et al. (2015), have been used to update the Ceit-IK4 PWM methodology to a new version of the methodology called Extended Plant-Wide Modelling (E-PWM). The modelling methodology developed for calculating heat variations caused by transformations has been validated with experimental and theoretical data obtained in literature. After validation, a verification of the predictive capacity of the heat transfer model was carried out by simulating the behaviour of an Autothermal Thermophilic Aerobic Digester (ATAD). In order to test the usefulness and applicability of the overall heat transfer model, two case studies have been carried out. In the first study, the same ATAD reactor was simulated, and in the second case study a global reference plant (BSM2) was used. In order to show the potential of the library, three evolutionary WWTPs were compared from a techno-economic standpoint. The selected configurations were (1) a conventional WWTP based on a modified version of the Benchmark Simulation Model No. 2, (2) an upgraded or retrofitted WWTP, and (3) a new Wastewater Resource Recovery Facility (WRRF) concept denominated as C/N/P decoupling WWTP. Subsequently, the thesis presents three case studies conducted in full-scale wastewater treatment plant in order to show the usefulness of adapted and flexible model libraries for optimising real full-scale WWTPs, as is the case of the PWM library. The thesis concludes with a chapter devoted to the most significant conclusions, the bibliography, and a set of appendixes with additional information, such as the detailed description of the models and the characterization of the components