Nuevas metodologías para el estudio de la interacción carbohidrato-proteína empleando la resonancia magnética nuclear

Tesis inédita de la Universidad Complutense de Madrid. Facultad de Farmacia, leída el 28-04-2020Carbohydrates (glycan, sugars, saccharides) play a key role in life and disease. Their wide presence in different processes comprises from the structural and energetic function to the basis of the molecular recognition of physiological and pathogenic events. Indeed, they are involved in many biological events of Paramount importance (i.e. cell-cell, cell-matrix or cell-molecules interactions). Therefore, the study of the molecular recognition events in which sugars are involved is a key task for understanding these interactions, and the structural elucidation and the conformational studies of saccharides are crucial to unravel their interaction mechanisms as well as the biological implications of these events. However, the inherent flexibility of carbohydrates precludes their crystallization, hampering the study by X-ray diffraction. In contrast, Nuclear Magnetic Resonance (NMR) permits to work in solution, mimicking the physiological conditions, and thus provides information on sugar geometries and dynamics. Therefore, NMR spectroscopy is the preferred technique for the structural characterization of carbohydrates, becoming a powerful tool for the understanding of the molecular processes in which carbohydrates are involved. In this context, two biological systems have been studied in this Thesis with the aim to understand the conformation and dynamics of different saccharides and glycomimetics in solution and to unravel key features of their molecular recognition processes with their receptors…Los carbohidratos (glicanos, azúcares, sacáridos) juegan un papel clave en la vida y en la enfermedad. Su amplia presencia en diferentes procesos abarca desde las funciones energética y estructural hasta las bases del reconocimiento molecular de eventos fisiológicos y patogénicos. De hecho, se encuentran involucrados en numerosos eventos biológicos de importancia (interacciones célula-célula, célulamatriz o célula-moléculas). Por lo tanto, el estudio de los eventos de reconocimiento molecular en los que los azúcares se encuentran involucrados es clave para entender estas interacciones, y la elucidación estructural y los estudios conformacionales son cruciales para descifrar sus mecanismos de interacción, así como las implicaciones biológicas de estos eventos. Sin embargo, la inherente flexibilidad de los carbohidratos impide su cristalización y por tanto su estudio por difracción de rayos X. Por el contrario, la Resonancia Magnética Nuclear (RMN) permite trabajar en disolución, imitando las condiciones fisiológicas , y por tanto, proporciona información sobre la geometría y la dinámica de los azúcares. Por tanto, la espectroscopía de RMN es la técnica preferida para la caracterización estructural de carbohidratos, siendo una técnica muy potente para entender los procesos moleculares en los que los carbohidratos están involucrados. En este contexto, se han estudiado en esta Tesis dos sistemas biológicos con el objetivo de entender la conformación y la dinámica de diferentes sacáridos y glicomiméticos en solución, y descifrar las características clave de sus procesos de reconocimiento molecular con sus receptores…Fac. de FarmaciaTRUEunpu

Enzymatic fine-tuning for 2-(6-hydroxynaphthyl) β-d-xylopyranoside synthesis catalyzed by the recombinant β-xylosidase BxTW1 from Talaromyces amestolkiae

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al.[Background]: Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal ß-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated. [Results]: The ß-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of ß-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-ß-d-xylopyranoside of 0.20 mM and 69.3 s¿1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) ß-d-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR. [Conclusions]: A ß-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) ß-d-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.This work was carried out with funding from projects BIO2015-68387-R, RTC-2014-1777-3 and CTQ2015-64597-C2 from MINECO and S2013/MAE2972 from Comunidad de Madrid, as well as from the Natural Sciences and Engineering Research Council of Canada. M. Nieto-Domínguez thanks the MINECO for an FPU fellowship.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

A glucotolerant β-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds

The interest for finding novel β-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. Results In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris, purified, and characterized. The enzyme showed good efficiency on p-nitrophenyl glucopyranoside (pNPG) (Km= 3.36 ± 0.7 mM, kcat= 898.31 s−1), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the β-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-d-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different pNP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG). Conclusions In this work, we report the characterization of a novel glucotolerant 1,2-β-glucosidase, which also has a considerable activity on 1,4-β-glucosyl bonds, that has been cloned in P. pastoris, produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications

16 derivas hipermínimas

Postprint (published version

Electrodeposited magnetic nanowires with radial modulation of composition

In the last few years, magnetic nanowires have gained attention due to their potential implementation as building blocks in spintronics applications and, in particular, in domain-wall- based devices. In these devices, the control of the magnetic properties is a must. Cylindrical magnetic nanowires can be synthesized rather easily by electrodeposition and the control of their magnetic properties can be achieved by modulating the composition of the nanowire along the axial direction. In this work, we report the possibility of introducing changes in the composition along the radial direction, increasing the degrees of freedom to harness the magnetization. In particular, we report the synthesis, using template-assisted deposition, of FeNi (or Co) magnetic nanowires, coated with a Au/Co (Au/FeNi) bilayer. The diameter of the nanowire as well as the thickness of both layers can be tuned at will. In addition to a detailed structural characterization, we report a preliminary study on the magnetic properties, establishing the role of each layer in the global collective behavior of the system

The Different Microbial Etiology of Prosthetic Joint Infections According to Route of Acquisition and Time After Prosthesis Implantation, Including the Role of Multidrug-Resistant Organisms

The aim of our study was to characterize the etiology of prosthetic joint infections (PJIs)-including multidrug-resistant organisms (MDRO)-by category of infection. A multicenter study of 2544 patients with PJIs was performed. We analyzed the causative microorganisms according to the Tsukayama's scheme (early postoperative, late chronic, and acute hematogenous infections (EPI, LCI, AHI) and "positive intraoperative cultures" (PIC)). Non-hematogenous PJIs were also evaluated according to time since surgery: 12 months. AHIs were mostly caused by Staphylococcus aureus (39.2%) and streptococci (30.2%). EPIs were characterized by a preponderance of virulent microorganisms (S. aureus, Gram-negative bacilli (GNB), enterococci), MDROs (24%) and polymicrobial infections (27.4%). Conversely, coagulase-negative staphylococci (CoNS) and Cutibacterium species were predominant in LCIs (54.5% and 6.1%, respectively) and PICs (57.1% and 15.1%). The percentage of MDROs isolated in EPIs was more than three times the percentage isolated in LCIs (7.8%) and more than twice the proportion found in AHI (10.9%). There was a significant decreasing linear trend over the four time intervals post-surgery for virulent microorganisms, MDROs, and polymicrobial infections, and a rising trend for CoNS, streptococci and Cutibacterium spp. The observed differences have important implications for the empirical antimicrobial treatment of PJIs.Acknowledgments: This work was supported by the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness (grant number PI15/1026) (Co-funded by European Regional Development Fund/European Social Fund "Investing in your future"). REIPI (Spanish Network for Research in Infectious Disease) is supported by the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness, and by the European Development Regional Fund “A way to achieve Europe”

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Evaluation of the linkage-disequilibrium method for the estimation of effective population size when generations overlap:an empirical case

Example of calculation of long-term contributions. (DOCX 14 kb