Consensus Design of an Evolved High-Redox Potential Laccase

Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.This study is based upon work funded by and the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016- 79106-R-Lignolution. BG-F was supported by a FPI national fellowship BES-2014-068887

Datasets on technological GHG emissions mitigation options for the agriculture sector

The 2030 EU policy framework for climate and energy confirms that all sectors, including agriculture, should contribute to climate stabilisation and greenhouse gas (GHG) emission reduction in the most cost-effective way. Since 2009, the European Commission's Joint Research Centre (JRC) analyses the economic impact of GHG mitigation policy options for EU agriculture. However, the lack of precise, integrated and harmonised data on the current and potential uptake, cost-effectiveness and GHG emissions reduction potential of technological (i.e. technical and management based) mitigation options hampers the analysis of the economic impacts of GHG mitigation in agriculture. Against this background, the JRC organised a workshop in Seville on 14th June 2016 which gathered European Commission staff and experts from diverse international institutions aiming to: i) identify current activities conducted by research institutes on the building of datasets for GHG mitigation technologies and their state and development, ii) establish synergies and working mechanisms among the different institutions working on the topic, iii) identify which are the current gaps and limitations of existing datasets and models and, iv) conceive a roadmap to build possible new datasets per mitigation technology. The present report is based on the workshop results and concludes on how to move forward.JRC.D.4-Economics of Agricultur

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

ABSTRACT Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back 500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3- cyclopentanedione oxidation, a –diketone initiator commonly used in vinyl polymerization reactions. IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the -diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers

Reliability and reproductibility of skin temperature of overweight subjects by an infrared thermograpy software designed for human beings

The technical improvement and new applications of Infrared Thermography (IRT) with healthy subjects should be accompanied by results about the reproducibility of IRT measurements in different popula-tion groups. In addition, there is a remarkable necessity of a larger supply on software to analyze IRT images of human beings. Therefore, the objectives of this study were: firstly, to investigate the reproducibility of skin temperature (Tsk) on overweight and obese subjects using IRT in different Regions of Interest (ROI), moments and side-to-side differences (?T); and secondly, to check the reliability of a new software called Termotracker®, specialized on the analysis of IRT images of human beings. Methods: 22 overweight and obese males (11) and females (11) (age: 41,51±7,76 years; height: 1,65±0,09 m; weight: 82,41±11,81 Kg; BMI: 30,17±2,58 kg/m²) were assessed in two consecutive thermograms (5 seconds in-between) by the same observer, using an infrared camera (FLIR T335, Sweden) to get 4 IRT images from the whole body. 11 ROI were selected using Termotracker® to analyze its reproducibility and reliability through Intra-class Correlation Coefficient (ICC) and Coefficient of Variation (CV) values. Results: The reproducibility of the side-to-side differences (?T) between two consecutive thermograms was very high in all ROIs (Mean ICC = 0,989), and excellent between two computers (Mean ICC = 0,998). The re-liability of the software was very high in all the ROIs (Mean ICC = 0,999). Intraexaminer reliability analysing the same subjects in two consecutive thermograms was also very high (Mean ICC = 0,997). CV values of the different ROIs were around 2%. Conclusions: Skin temperature on overweight subjects had an excellent reproducibility for consecutive ther-mograms. The reproducibility of thermal asymmetries (?T) was also good but it had the influence of several factors that should be further investigated. Termotracker® reached excellent reliability results and it is a relia-ble and objective software to analyse IRT images of humans beings

Thyroid stimulating hormone levels and geriatric syndromes : secondary nested case–control study of the Mexican Health and Aging Study

Q3Q3Abstract Purpose To determine the incidence of geriatric syndromes (GS) in community dwelling older adults with subclinical hypothyroidism. Methods This is an analysis from the Mexican Health and Aging Study, of a subsample of 2089 subjects with TSH determination. From this last subsample, we included 1628 individuals with TSH levels in the subclinical range (4.5–10 µU/ml). Results The multivariate analysis showed that when comparing data obtained from the 2012 wave with the 2015 wave results, there was a signifcant incidence of some GS such as falls (OR 1.79, CI 1.16–2.77, p=0.0116), fatigue (OR 2.17, CI 1.40–3.38, p=0.0348) and depression (OR 1.70, CI 1.06–2.71, p=0.0246) among the subclinical hypothyroidism group. Conclusion This study showed a greater incidence of GS in subjects 50 years and older with sub-clinical hypothyroidism, when compared to those with normal thyroid function. Keywords Thyroid stimulating hormone · Aging · Geriatric syndromes · Chronic disease · Subclinical hypothyroidismhttps://orcid.org/0000-0002-1652-5042https://scholar.google.com/citations?user=qUwLuswAAAAJ&hl=es&oi=aohttps://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000136038Revista Internacional - Indexad

Farmacoalegría

Sección Deptal. de Historia del Arte III (Contemporáneo)Fac. de Bellas ArtesFALSEsubmitte

Impact of sarcopenia and frailty in a multicenter cohort of polypathological patients

The prevalence, relationships and outcomes of sarcopenia and frailty in polypathological patients remain unknown. We performed a multicenter prospective observational study in six hospitals in order to assess prevalence, clinical features, outcome and associated risk factors of sarcopenia and frailty in a hospital-based population of polypathological patients. The cohort was recruited by performing prevalence surveys every 14 days during the inclusion period (March 2012-June 2016). Sarcopenia was assessed by means of EWGSOP criteria and frailty by means of Fried''s criteria. Skeletal muscle mass was measured by tetrapolar bioimpedanciometry. All patients were followed for 12 months. Factors associated with sarcopenia, frailty and mortality were analyzed by multivariate logistic regression, and Kaplan-Meier curves. A total of 444 patients (77.3 +/- 8.4 years, 55% males) were included. Sarcopenia was present in 97 patients (21.8%), this being moderate in 54 (12.2%), and severe in 43 (9.6%); frailty was present in 278 patients (62.6%), and 140 (31.6%) were pre-frail; combined sarcopenia and frailty were present in the same patient in 80 (18%) patients. Factors independently associated to the presence of both, sarcopenia and frailty were female gender, older age, different chronic conditions, poor functional status, low body mass index, asthenia and depressive disorders, and low leucocytes and lymphocytes count. Mortality in the 12-months follow-up period was 40%. Patients with sarcopenia, frailty or both survived significantly less than those without these conditions. Sarcopenia and frailty are frequent and interrelated conditions in polypathological patients, shadowing their survival. Their early recognition and management could improve health-related outcomes in this population

Circulating omentin concentration increases after weight loss

Omentin-1 is a novel adipokine expressed in visceral adipose tissue and negatively associated with insulin resistance and obesity. We aimed to study the effects of weight loss-induced improved insulin sensitivity on circulating omentin concentrations. METHODS: Circulating omentin-1 (ELISA) concentration in association with metabolic variables was measured in 35 obese subjects (18 men, 17 women) before and after hypocaloric weight loss. RESULTS: Baseline circulating omentin-1 concentrations correlated negatively with BMI (r = -0.58, p < 0.001), body weight (r = -0.35, p = 0.045), fat mass (r = -0.67, p < 0.001), circulating leptin (r = -0.7, p < 0.001) and fasting insulin (r = -0.37, p = 0.03). Circulating omentin-1 concentration increased significantly after weight loss (from 44.9 +/- 9.02 to 53.41 +/- 8.8 ng/ml, p < 0.001). This increase in circulating omentin after weight loss was associated with improved insulin sensitivity (negatively associated with HOMA value and fasting insulin, r = -0.42, p = 0.02 and r = -0.45, p = 0.01, respectively) and decreased BMI (r = -0.54, p = 0.001). CONCLUSION: As previously described with adiponectin, circulating omentin-1 concentrations increase after weight loss-induced improvement of insulin sensitivity

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.This manuscript was funded by grants PDI-2020-120412RB-I00 and PDC2021- 121719-I00 (FS-M) and PID2020- 119352RB-I00 (AS) from the Spanish Ministry of Economy and Competitiveness; CAM (S2017/BMD3671-INFLAMUNE-CM) from the Comunidad de Madrid (FS-M). CIBERCV (CB16/11/00272) and BIOIMID PIE13/041 from the Instituto de Salud Carlos. The current research has received funding from 'la Caixa' Foundation under the project code HR17-00016. Grants from Ramón Areces Foundation 'Ciencias de la Vida y de la Salud' (XIX Concurso-2018) and from Ayuda Fundación BBVA y Equipo de Investigación Científica (BIOMEDICINA-2018) (to FSM). The CNIC is supported by the Ministerio de Ciencia, Innovacion y Universidades and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015–0505). IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, CEX2020-001039-S). SGD is supported by a grant from the Spanish Ministry of Universities. Authors thank Dr Miguel Vicente-Manzanares for critical review and editing. We also thank Dr Francisco Urbano and Dr Covadonga Aguado for their support with EM (TEM facilities, Universidad Autónoma de Madrid).S