Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.This work was supported by grants from the Spanish Ministry of Science and Innovation (https://www.ciencia.gob.es/) with FEDER funds (RTI2018- 099267- B- I00) and a Tatiana Foundation project (P-048-FTPGB 2018) to AS. VS-T holds a predoctoral fellowship from the Basque Government

Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types

Aproximación al estudio de la relación mujer-economía en los libros de texto de la España democrática : análisis comparativo entre el periodo de la transición y los primeros años del siglo XXI

La relación entre mujer y cultura económica en el panorama español ha sido poco estudiada por los historiadores, mucho menos si la vinculamos a la socialización recibida durante los años de formación escolar obligatoria, que creemos que influyen en la futura elección profesional. Los libros de texto, recursos didácticos muy utilizados por el profesorado, pueden proporcionar importantes pistas acerca de la toma de decisiones de las mujeres con relación a las actividades que van a desempeñar dentro del campo económico, puesto que reflejan los estereotipos e ideologías que caracterizan la mentalidad dominante de una determinada época y sociedad. Este será el objetivo de nuestro trabajo: observar el papel de las mujeres respecto al ámbito económico, mediante un análisis de contenido tanto de las imágenes como de los textos escritos de manuales utilizados en la etapa de obligatoriedad escolar comparando dos períodos: la Transición y los primeros años de la democracia.Historians have not deeply explored the relationship between women and economic culture in the Spanish panorama, much less, if we link it to the socialization received during compulsory schooling years which influences future career choices. Textbooks have dominated over other resources at school and they can provide important clues about women's decision-making in relation to their economic activities, as they incorporated the underpinnings of social, cultural and economic mainstream. Likewise, we think that sexist stereotypes abound in textbooks too. This paper presents a content analysis of textbooks, both of the illustrations and the text itself. The aim of this research is to observe the economic role of women rendered in primary studies textbooks after Franco’s regime, comparing those used during the early years of democracy, also called “Transición” and nowaday’s Spain

Identidad profesional y socialización de género: un estudio desde la manualística escolar en la España democrática

The construction of professional identity is a concept that holds interest for a variety of disciplines such as Anthropology, Psychology or Pedagogy. From the perspective of social constructivism, it has a narrative, dynamic character and is shaped by the interaction of the subject with current socio-historical discourses and their interiorization. During the decade of the nineteen seventies, the labor market in Spain experienced a massive incorporation of women, brought about by social and legislative advances in education and employment. The years of Spanish democracy are essential in the configuration and renewal of female identity in the economic field. This article focuses on the influence of the school textbook - the main didactic resource used in formal education - in this configuration. It presents models and stereotypes linked to gender, with a potential influence on the transmission and configuration of a biased socio-labor culture. In order to study this influence,  our research examines a total of 20 elementary education textbooks in the areas of Language and Social Sciences in two periods of democratic Spain —the so-called Transition period and the current moment—, to explore the professional identity of women linked to the economic socialization as this is conveyed through this educational tool. Our results reveal a minimal inclusion of women as economic subjects in the textbooks; the underrepresentation of females  performing paid activities, as well the lack of professional diversity serve to highlight a substantial inequality in the socialization processes between men and women, contrary to the goals of  coeducational teaching values. La construcción de la identidad profesional es un tema de interés para diferentes disciplinas como la antropología, la psicología o la educación. Siguiendo el enfoque del constructivismo social, entendemos que esta tiene un carácter narrativo y dinámico y que se forma a partir de la interiorización de discursos sociohistóricos vigentes en las situaciones con las que el sujeto interacciona. Durante la década de los años setenta del siglo XX, el mercado laboral en España experimentó una masiva incorporación de las mujeres, promovida por progresos sociales y legislativos en educación y empleo. Los años de democracia española han resultado fundamentales en la configuración y renovación de la identidad femenina en el ámbito económico. El manual escolar, principal recurso didáctico utilizado en la educación formal, se presenta como un instrumento socializador con poder relevante en la transmisión y configuración de una cultura sociolaboral sesgada en caso de contener estereotipos de género. Nuestra investigación examina un total de veinte libros de texto de Educación Primaria de las áreas de Lengua y Sociales en dos periodos de la España democrática - Transición política y actualidad -, estudiando la identidad profesional femenina vinculada a la socialización económica difundida mediante este material. Los resultados desvelan una escasa participación de las mujeres como sujetos económicos en los manuales, mostrando una insuficiente representación femenina en el desempeño de actividades remuneradas, así como una falta de diversidad profesional que pone en relieve una gran desigualdad en los procesos de socialización entre hombres y mujeres contrarios a valores coeducativos.

Economía y mujer en la manualística escolar de la España democrática: análisis comparativo entre el período de la Transición y los primeros años del siglo XXI

A lo largo de los años, los manuales escolares han sido uno de los recursos didácticos más utilizados por el profesorado. El libro escolar es, además de un soporte curricular a través del cual se transmite el conocimiento académico que las instituciones han de propagar, un reflejo de la sociedad que lo produce en cuanto a que en él se vehiculan valores, actitudes, estereotipos e ideologías que caracterizan la mentalidad dominante de una determinada época o, lo que es lo mismo, el imaginario colectivo que configura el currículum explícito, y también el oculto. Dicho imaginario ha sostenido la persistencia de una sociedad patriarcal, en la que la mujer aparece en un lugar secundario con respecto al hombre, y con unos roles prefijados conectados a una visión esencialista de la condición femenina. Cabría pensar que España, tras el fin de la dictadura franquista, ha experimentado una evolución significativa en la forma de concebir las funciones políticas, económicas y sociales adjudicadas a ambos sexos, intentando evitar desigualdades perniciosas. Dicha evolución debería verse reflejada en los libros de texto que el alumnado maneja en las aulas. A través de este trabajo, y mediante análisis tanto cuantitativo como cualitativo tanto de las imágenes como de los textos verbales, queremos observar si dicha evolución ha existido, sobre todo desde el punto de vista de la denominada «cultura económica», analizando manuales escolares utilizados en la etapa de obligatoriedad escolar en dos períodos: la Transición y los primeros años del siglo XXI. Se trata de establecer cambios y permanencias, lo que nos permitirá dar recomendaciones de cara a la confección y/o uso de e ste tipo de recurso en la escuela.Over the years, textbooks have dominated over other resources at school. Textbooks are also a conduit for the academic content that institutions want to deliver, a reflection of society in that the mainstream social attitudes, values and stereotypes and ideologies are reflected in their pages. It is a well-known fact that the established mindset is disseminated with them, hence shaping an overt and covert curriculum. Textbooks therefore reproduce the collective imagination dominated by a patriarchal society, one in which women play a second position and play traditional female roles. Altogether, it paints the essentialist female condition. Post-Franco Spain put an end to dictatorial manners and now new social, economic and political reality should prevail and harmful sex inequality should have vanished too. Textbooks should replicate this new lifestyle. This paper presents a content analysis of textbooks, both of the illustrations and the text itself. We want to test if this change has taken place a tall, with a particular analysis of the economic culture. We therefore compare textbooks of the time right after the dictatorship, some forty years ago with the textbooks in use now at the turn of the century. We explore differences and similarities which may inspire new textbooks for the present time

Microglial phagocytosis dysfunction in the dentate gyrus is related to local neuronal activity in a genetic model of epilepsy

Objective Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very efficient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB). Methods We used confocal imaging and stereology-based quantification of apoptosis and phagocytosis of the hippocampus ofCstbknockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduceCstbexpression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apoptosis, phagocytosis, and active hippocampal neurons. Results Microglial phagocytosis was impaired in the hippocampus ofCstbKO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present inCstbKO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) inCstb-deficient mice were at close proximity to active cFos(+)neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos(+)neurons was specific forCstbKO mice. Significance These results suggest a complex crosstalk between apoptosis, phagocytosis, and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction inCstbKO mice. Overall, these data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.Peer 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

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries