Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Device-independent quantum key distribution with realistic single-photon source implementations.

Device-independent quantum key distribution (DIQKD) aims at generating secret keys between distant parties without the parties trusting their devices. We investigate a proposal for performing fully photonic DIQKD, based on single photon sources and heralding measurements at a central station placed between the two parties. We derive conditions to attain non-zero secret-key rates in terms of the photon efficiency, indistinguishability and the second order autocorrelation function of the single-photon sources. Exploiting new results on the security bound of such protocols allows us to reduce the requirements on the physical parameters of the setup. Our analysis shows that in the considered schemes, key rates of several hundreds of secret bits per second are within reach at distances of several tens of kilometers.

[Educational interventions in childhood asthma: A systematic review]. / Intervenciones educativas en asma infantil: Una revisión sistemática.

OBJECTIVE: To analyze educational interventions in pediatric asthmatic patients to achieve an adequate inhalation technique and improve their self-management. DESIGN: Systematic review based on the PRISMA recommendations. DATA SOURCES: Pubmed, Scopus, Cuiden, Web of Science and Google Scholar databases were reviewed. STUDY SELECTION: Sixteen articles published between 2014 and 2021 were included, with access to full text, languages: English, French and Spanish and pediatric population: 0-18 years. DATA EXTRACTION: Two thousand three hundred and thirteen children were participated. The variables analyzed were: level of care, type of intervention, correct performance of the inhalation technique, follow-up of the technique, delivery of written recommendations, professional-educator category, variables related to respiratory pathology, school absenteeism, quality of life and economic costs. RESULTS: The health care level was primary, hospital and community care, where specialist doctors, nurses and pharmacists stood out as educators. The most prevalent educational interventions are on-site demonstration and delivery of recommendations or multimedia interventions. Several articles report that asthma education is not carried out correctly, others state that their technique improves after the intervention, but most of them highlight the importance of periodic review of the technique. CONCLUSIONS: The authors report improvement in the inhalation technique in all of them, as well as greater self-management of the disease and adherence to treatment. It is necessary to intensify the education of patients in the correct handling of the devices, and the follow-up and subsequent review to optimize the control of the disease.

Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

MiR-9-5p protects from kidney fibrosis by metabolic reprogramming.

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally and control biological processes (BPs), including fibrogenesis. Kidney fibrosis remains a clinical challenge and miRNAs may represent a valid therapeutic avenue. We show that miR-9-5p protected from renal fibrosis in the mouse model of unilateral ureteral obstruction (UUO). This was reflected in reduced expression of pro-fibrotic markers, decreased number of infiltrating monocytes/macrophages, and diminished tubular epithelial cell injury and transforming growth factor-beta 1 (TGF-ß1)-dependent de-differentiation in human kidney proximal tubular (HKC-8) cells. RNA-sequencing (RNA-Seq) studies in the UUO model revealed that treatment with miR-9-5p prevented the downregulation of genes related to key metabolic pathways, including mitochondrial function, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and glycolysis. Studies in human tubular epithelial cells demonstrated that miR-9-5p impeded TGF-ß1-induced bioenergetics derangement. The expression of the FAO-related axis peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-peroxisome proliferator-activated receptor alpha (PPARα) was reduced by UUO, although preserved by the administration of miR-9-5p. We found that in mice null for the mitochondrial master regulator PGC-1α, miR-9-5p was unable to promote a protective effect in the UUO model. We propose that miR-9-5p elicits a protective response to chronic kidney injury and renal fibrosis by inducing reprogramming of the metabolic derangement and mitochondrial dysfunction affecting tubular epithelial cells.

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice.

Neuroinflammation and microglial activation are significant processes in Alzheimer's disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer's disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer's disease and other tau-mediated neurodegenerative diseases.

Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae.

The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.

Continuous versus discontinuous suture in perineal injuries produced during delivery in primiparous women: a randomized controlled trial.

BACKGROUND: The technique used in the repair of a perineal injury resulting from childbirth could avoid discomfort and morbidity during the postpartum period. Recent studies show inconsistent results and support the need for new research with the inclusion of new health parameters not yet studied. Therefore, this study aims to evaluate if the suture technique (continuous or interrupted) has an effect on pain and other postpartum problems, incidence of incontinence (urinary and/or fecal), and the restart of sexual relations. METHODS: A single-blind randomized clinical trial was conducted in five hospitals in south-east Spain. The participants were primiparous women who had experienced a perineal injury during delivery (second-degree tear or episiotomy). Data was collected on sociodemographic variables, variables associated with pregnancy, labor and delivery, and the postpartum period, and outcomes during the 3 months after delivery: pain, incontinence, and restart of sexual relations. Odds ratios (OR) were calculated by binary logistic regression to assess the influence of the suture type on binary outcomes and t-test used for comparing continuous outcomes. Multivariate analyses (using logistic regression -adjusted (aOR)- and analysis of covariance) were carried out to adjust for unbalanced variables after randomization. RESULTS: A total of 70 women were included in the intervention group (continuous suture) and 64 in the reference group (interrupted sutures). A negative association was observed (aOR = 0.39; 95% CI = 0.18-0.86) between a continuous suture and the need for analgesia at 24 h postpartum. Pain experienced by the women at 24 h postpartum was assessed as 4.4 ± 0.3 compared with a score of 3.4 ± 0.3 in the group with continuous sutures (p = 0.011). At 15 days postpartum, women in the intervention group experienced less pain (aOR = 0.38; 95% CI = 0.18-0.80) (p = 0.019). Urinary sphincter incontinence was also evaluated at 15 days, with 4.3% (n = 3) of the women in the intervention group presenting with urinary incontinence compared with 18.8% (n = 12) in the control group (aOR = 0.11; 95% CI = 0.03-0.47) (P = 0.003). CONCLUSIONS: The women who had a continuous suture repair showed lower levels of pain from delivery to 3 months after delivery and had a lower incidence of urinary incontinence at 15 days postpartum. TRIAL REGISTRATION: ClinicalTrials.gov NCT03825211 posted January 31, 2019 (retrospectively registered).

Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase.

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.

Shuffling the Neutral Drift of Unspecific Peroxygenase in Saccharomyces cerevisiae.

Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae, so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivoIMPORTANCE Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae.

miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2.

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-ß1 (TGF-ß1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-ß receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-ß1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-ß1 induces miR-9-5p expression as a self-limiting homeostatic response.

Unveiling the basis of alkaline stability of an evolved versatile peroxidase.

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441: , 487-498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability.

Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.

AIM: To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources. METHODS: An e-mail survey of 257 CESEE key informants involved in HTA was undertaken between March and April 2014. Contact e-mail addresses were identified from the internet. The survey questionnaire consisted of 3 sections: i) characteristics of the organization performing HTA, (ii) networking in HTA, and (iii) resources allocated for HTA. RESULTS: The survey was completed by 41 respondents representing a wide range of institutions from CESEE countries (response rate of 19.8%). Less than a quarter of respondents reported that their institutions had HTA-specific budgets, whereas the majority indicated that their institutions participated in HTA networks either at domestic or international levels. Although almost half of respondents indicated that their institutions offered HTA training, a shortage in skills training was suggested as the main barrier to HTA. CONCLUSION: This is the first survey to thoroughly assess the state of HTA capacity in the CESEE region. To strengthen HTA capacity, CESEE countries should increase financial, technical, and training resources. To strengthen collaboration, the European Union and other international bodies should assist existing HTA networks in fulfilling their regional activities through leadership, advocacy to local policymakers, funding, and technical assistance.

The effect of fatigue and fibromyalgia on sexual dysfunction in women with chronic fatigue syndrome.

Sexual dysfunction in patients with chronic fatigue syndrome is attracting growing interest but, to date, few studies have analyzed it. For this reason, the authors evaluated sexual dysfunction in women with chronic fatigue syndrome (using the Golombok Rust Inventory of Sexual Satisfaction) and explore correlations with fatigue and other symptoms. Sexual dysfunction was greater in patients with chronic fatigue syndrome (n = 615) with a higher number of cognitive, neurological, and neurovegetative symptoms, concomitant fibromyalgia, Sjögren's syndrome, or myofascial pain syndrome, and more intense fatigue (p <.05).

Intrapleural Fibrinolysis with Urokinase Versus Alteplase in Complicated Parapneumonic Pleural Effusions and Empyemas: A Prospective Randomized Study.

BACKGROUND: Pleurofibrinolysis has been reported to be potentially beneficial in the management of complicated parapneumonic effusions (CPPE) and empyemas in the adult population. METHODS: Prospective, controlled, randomized, and double-blind study, to evaluate intrapleural alteplase 10 mg (initially 20 mg was considered but bleeding events forced dose reduction) versus 100,000 UI urokinase every 24 h for a maximum of 6 days in patients with CPPE or empyemas. The primary aim was to evaluate the success rate of each fibrinolytic agent at 3 and 6 days. Success of therapy was defined as the presence of both clinical and radiological improvement, making additional fibrinolytic doses unnecessary, and eventually leading to resolution. Secondary outcomes included the safety profile of intrapleural fibrinolytics, referral for surgery, length of hospital stay, and mortality. RESULTS: A total of 99 patients were included, of whom 51 received alteplase and 48 urokinase. Success rates for urokinase and alteplase at 3 and 6 days were not significantly different, but when only the subgroup of CPPE was considered, urokinase resulted in a high proportion of cures. There were no differences in mortality or surgical need (overall, 3 %). Five (28 %) patients receiving 20 mg of alteplase and 4 (12 %) receiving 10 mg presented serious bleeding events. CONCLUSIONS: If intrapleural fibrinolytics are intended to be used, urokinase may be more effective than alteplase in patients with non-purulent CPPE and have a lower rate of adverse events.

Directed evolution of unspecific peroxygenase from Agrocybe aegerita.

Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase.

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.

[Comparison of Heart, Grace Scores and clinical parameters as predictors of cardiovascular events in patients with chest pain in the Emergency Department]. / Comparacion de las escalas Heart, Grace Score y los parámetros clínicos como predictores de eventos cardiovasculares en pacientes con dolor torácico en Urgencias.

OBJECTIVE: Risk assessment of patients with chest pain is based on clinical parameters; however, without a scoring system, such as risk stratification scales, estimates are less precise and accurate. The aim of this paper was to compare the HEART, GRACE score and clinical parameters in the prediction of major cardiovascular events (cardiovascular mortality or acute myocardial infarction) during hospitalization, in patients with chest pain attended in the emergency department. METHODS: A descriptive observational study of patients with ischemic chest pain, who attended to the Miguel Servet University Hospital emergency department (Zaragoza, Spain) during one year was carried out. HEART and GRACE scores were calculated retrospectively from clinical history. Quantitative variables were expressed as mean (±standard deviation), and qualitative variables as frequencies and percentages. A bivariate analysis was carried out using the chi-square test. The performance of the scales and clinical parameters was compared by calculating the area under the curve. The primary outcome was the occurrence of a major cardiovascular event (cardiovascular mortality or acute myocardial infarction) during hospital admission. RESULTS: 306 patients were registered (66.3% men, n=203), with a mean age of 71.45±12.85 years and a 48.7% history of ischemic heart disease. The areas under the curve for HEART scales, GRACE and clinical parameters were 0.80 (95% CI: 0.73-0.86), 0.79 (95% CI: 0.72-0.85) and 0.74 (95% CI: 0.68-0.80), respectively. During hospitalization, the incidence of the primary event was 13.4% and no low-risk patient, in both scales, presented a major cardiovascular event. CONCLUSIONS: In patients with ischemic chest pain attended in the emergency department, the GRACE and HEART scale have a greater area under curve than clinical parameters.

OBJECTIVE: La valoración del riesgo de los pacientes con dolor torácico se basa en los parámetros clínicos. Sin embargo, sin un sistema de puntuación, como las escalas de estratificación del riesgo, las estimaciones son menos precisas y exactas. El objetivo de este estudio fue comparar las escalas HEART, GRACE Score y los parámetros clínicos en la predicción de eventos mayores cardiovasculares (mortalidad cardiovascular o infarto agudo de miocardio) durante la hospitalización, en pacientes con dolor torácico atendidos en Urgencias. METHODS: Se realizó un estudio observacional descriptivo de pacientes que, durante un año, acudieron a Urgencias del Hospital Universitario Miguel Servet (Zaragoza) por dolor torácico de tipo isquémico. Las puntuaciones HEART y GRACE se calcularon retrospectivamente a partir de las historias clínicas. Las variables cuantitativas se expresaron como media (±desviación estándar), y las cualitativas como frecuencias y porcentajes. Se llevó a cabo un análisis bivariante mediante la prueba chi cuadrado. El rendimiento de las escalas y parámetros clínicos se comparó mediante el cálculo del área bajo la curva. El resultado primario fue la ocurrencia de un evento mayor cardiovascular (mortalidad cardiovascular o infarto agudo de miocardio) durante el ingreso hospitalario. RESULTS: Se registraron 306 pacientes (66,3% eran hombres, n=203), con edad media de 71,45±12,85 años y un 48,7% de antecedentes de cardiopatía isquémica. El área bajo la curva, para el evento primario, de las escalas HEART, GRACE y parámetros clínicos fue 0,80 (IC al 95%: 0,73-0,86), 0,79 (IC al 95%: 0,72- 0,85) y 0,74 (IC del 95%: 0,68-0,80), respectivamente. Durante la hospitalización, la incidencia del evento primario fue del 13,4% y ningún paciente de bajo riesgo, en ambas escalas, presentó un evento mayor cardiovascular. CONCLUSIONS: En pacientes con dolor torácico de tipo isquémico atendidos en Urgencias, tanto la escala GRACE como la escala HEART presentan un área bajo la curva más alta que los parámetros clínicos.

Tau-mediated axonal degeneration is prevented by activation of the WldS pathway.

Tauopathy is characterized by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule-associated protein tau. The neuronal changes evident in tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration. The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (WldS) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by co-expression of WldS. In a Drosophila model of tauopathy in which expression of human 0N3R tau protein leads to progressive age-dependent phenotypes, WldS was expressed with and without activation of the downstream pathway. The olfactory receptor neuron circuit OR47b was used for these studies in adults, and the larval motor neuron system was employed in larvae. Tau phenotypes studied included neurodegeneration, axonal transport, synaptic deficits and locomotor behaviour. Impact on total tau was ascertained by assessing total, phosphorylated and misfolded tau levels by immunohistochemistry. Activation of the pathway downstream of WldS completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of WldS was activated several weeks after tau-mediated degeneration had become established. Though total tau levels were not altered, the protected neurons displayed significantly reduced MC1 immunoreactivity suggestive of clearance of misfolded tau, as well as a trend for a decline in tau species phosphorylated at the AT8 and PHF1 epitopes. In contrast, WldS expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in tau-expressing larvae. This collectively implies that the pathway mediating the protective effect of WldS intersects with the mechanism(s) of degeneration initiated by tau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.

Age-related changes in tau and autophagy in human brain in the absence of neurodegeneration.

Tau becomes abnormally hyper-phosphorylated and aggregated in tauopathies like Alzheimers disease (AD). As age is the greatest risk factor for developing AD, it is important to understand how tau protein itself, and the pathways implicated in its turnover, change during aging. We investigated age-related changes in total and phosphorylated tau in brain samples from two cohorts of cognitively normal individuals spanning 19-74 years, without overt neurodegeneration. One cohort utilised resected tissue and the other used post-mortem tissue. Total soluble tau levels declined with age in both cohorts. Phosphorylated tau was undetectable in the post-mortem tissue but was clearly evident in the resected tissue and did not undergo significant age-related change. To ascertain if the decline in soluble tau was correlated with age-related changes in autophagy, three markers of autophagy were tested but only two appeared to increase with age and the third was unchanged. This implies that in individuals who do not develop neurodegeneration, there is an age-related reduction in soluble tau which could potentially be due to age-related changes in autophagy. Thus, to explore how an age-related increase in autophagy might influence tau-mediated dysfunctions in vivo, autophagy was enhanced in a Drosophila model and all age-related tau phenotypes were significantly ameliorated. These data shed light on age-related physiological changes in proteins implicated in AD and highlights the need to study pathways that may be responsible for these changes. It also demonstrates the therapeutic potential of interventions that upregulate turnover of aggregate-prone proteins during aging.