Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres

[EN] Hydrogen peroxide (H2O2) is generated in cells and plays an important role as a signalling molecule. It has been reported that H2O2 is involved in physiological and pathological processes in skeletal muscle. However, H2O2 detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H2O2 specifcally in real time using fuorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in diferent models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fbres isolated from C57BL/6J mice, and to measure intracellular H2O2 in real time in these cells. The results show that the expression of the HyPer biosensor in skeletal muscle cells is possible. In addition, we demonstrate that HyPer is functional and that this biosensor detects changes and fuctuations in intracellular H2O2 in a reversible manner. The HyPer2 biosensor, which is a more advanced version of HyPer, presents improved properties in terms of sensitivity in detecting lower concentrations of H2O2 in skeletal muscle fbres. In conclusion, the expression of the HyPer biosensor in the diferent experimental models combined with fuorescence microscopy techniques is a powerful methodology to monitor and register intracellular H2O2 specifcally in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H2O2 in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specifc biosensors for diferent reactive oxygen and nitrogen species or metabolites involved in cellular functions

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

The Use of Corticosteroids or Tocilizumab in COVID-19 Based on Inflammatory Markers

SEMI-COVID-19 Network.[Background] The inflammatory cascade is the main cause of death in COVID-19 patients. Corticosteroids (CS) and tocilizumab (TCZ) are available to treat this escalation but which patients to administer it remains undefined.[Objective] We aimed to evaluate the efficacy of immunosuppressive/anti-inflammatory therapy in COVID-19, based on the degree of inflammation. .[Design] A retrospective cohort study with data on patients collected and followed up from March 1st, 2020, to May 1st, 2021, from the nationwide Spanish SEMI-COVID-19 Registry. Patients under treatment with CS vs. those under CS plus TCZ were compared. Effectiveness was explored in 3 risk categories (low, intermediate, high) based on lymphocyte count, C-reactive protein (CRP), lactate dehydrogenase (LDH), ferritin, and d-dimer values.[Patients] A total of 21,962 patients were included in the Registry by May 2021. Of these, 5940 met the inclusion criteria for the present study (5332 were treated with CS and 608 with CS plus TCZ).[Main Measures] The primary outcome of the study was in-hospital mortality. Secondary outcomes were the composite variable of in-hospital mortality, requirement for high-flow nasal cannula (HFNC), non-invasive mechanical ventilation (NIMV), invasive mechanical ventilation (IMV), or intensive care unit (ICU) admission.[Key Results] A total of 5940 met the inclusion criteria for the present study (5332 were treated with CS and 608 with CS plus TCZ). No significant differences were observed in either the low/intermediate-risk category (1.5% vs. 7.4%, p=0.175) or the high-risk category (23.1% vs. 20%, p=0.223) after propensity score matching. A statistically significant lower mortality was observed in the very high–risk category (31.9% vs. 23.9%, p=0.049).[Conclusions] The prescription of CS alone or in combination with TCZ should be based on the degrees of inflammation and reserve the CS plus TCZ combination for patients at high and especially very high risk.Peer reviewe

Resultados clínicos iniciales y variables pronósticas en la implementación de un Código Sepsis en un Hospital Universitario de alta complejidad

Objective: To assess the impact of the first months of application of a Code Sepsis in a high complexity hospital, analyzing patient´s epidemiological and clinical characteristics and prognostic factors. Methods: A long-term observational study was carried out throughout a consecutive period of seven months (February 2015 - September 2015). The relationship with mortality of risk factors, and analytic values was analyzed using uni- and multivariate analyses. Results: A total of 237 patients were included. The in-hospital mortality was 24% at 30 days and 27% at 60 days. The mortality of patients admitted to Critical Care Units was 30%. Significant differences were found between the patients who died and those who survived in mean levels of creatinine (2.30 vs 1.46 mg/dL, p 65 years (OR 5.33, p 3 mmol/L (OR 5,85, p 1,2 mgr /dL (OR 4,54, p <0,05) and shock (OR 6,57, P <0,05). Conclusions: The epidemiological, clinical and mortality characteristics of the patients in our series are similar to the best published in the literature. The study has identified several markers that could be useful at a local level to estimate risk of death in septic patients. Studies like this one are necessary to make improvements in the Code Sepsis programsObjetivo. Evaluar el impacto de un programa educativo y organizativo llamado Código Sepsis, en los primeros siete meses de su aplicación en un hospital de alta complejidad. Material y métodos. Se realizó un estudio observacional durante un período consecutivo de siete meses (Febrero 2015-Septiembre 2015). Se analizó la relación con la mortalidad de los factores de riesgo y los valores analíticos usando análisis uni y multivariante. Resultados. Se incluyeron un total de 237 pacientes. La mortalidad intrahospitalaria a los 30 días fue del 24 % y del 27% a los 60 días. La mortalidad de los pacientes ingresados en Unidades de Cuidados Críticos fue del 30%. Se encontraron diferencias significativas entre los pacientes que murieron y los que sobrevivieron en sus valores medios de creatinina (2,30 vs 1,46 mg/dL, p <0,05), ácido láctico (6,10 vs 2,62 mmol/L, p <0,05) y procalcitonina (23,27 vs 12,73 mg/dL, p <0,05). Se encontró una tendencia lineal estadísticamente significativa entre los valores de la escala SOFA y la mortalidad (p <0,05). En el análisis multivariante se identificaron otros factores de riesgo independientes asociados con la muerte: edad > 65 años (OR 5,33, p 3 mmol/L (OR 5,85, p <0,05), creatinina > 1,2 mgr/dL (OR 4,54, p <0,05) y el shock (OR 6,57, P <0,05). Conclusiones. La mortalidad en este estudio se encuentra dentro de los límites de los ensayos clínicos más recientes de sepsis. El estudio ha identificado varios marcadores que podrían ser útiles a nivel local para estimar el riesgo en pacientes sépticos. Estudios como éste son necesarios para hacer mejoras en los programas de Código Sepsi

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

La ciudad como experiencia de conocimiento: Nuevas metodologías de aprendizaje, iniciación de la investigación y extensión universitaria a partir de la historia urbana

Depto. de GeografíaDepto. de Historia Moderna y ContemporáneaDepto. de Historia del ArteFac. de Geografía e HistoriaFALSEsubmitte