Optimization of brain organoids as models for the study of neurodegenerative diseases

ABSTRACTMotivation: One of the current challenges faced by neuroscience is the limited availability of in vitro models of neurodegenerative diseases, as there are notable anatomical and molecular differences among murine and human brain. As a result, significant efforts have been made towards the development of new models based on human cells, with cerebral organoids standing out as a particularly promising approach. Brain organoids are 3D models usually developed from induced pluripotent stem cells (iPSCs) that simulate the composition and cytoarchitecture of different regions of the human brain. They may allow us to obtain in vitro information about the human brain, which makes them a valuable model for investigating neurodegenerative diseases. Nevertheless, they present disadvantages associated with the absence of essential components for their development and functionality. Therefore, we will investigate the effect of incorporating an extracellular matrix (ECM) from human and pig brain into the culture of these organoids, as it contains specific combinations of components that play a role in multiple neuronal processes. On the other hand, in order to find the optimal model for generating this organoids, two protocols, Lancaster (1) and Rosebrock (2), have been compared. Lancaster’s protocol is the most cited for brain organoids, but following it, other embryonic layers are developed. Rosebrock’s protocol, is a modification of the Lancaster’s protocol, in which SMAD pathway inhibitors are used to avoid the formation of non-ectodermal layers.Methods: We generated brain organoids from iPSC, following two protocols: Lancaster (1) and Rosebrock (2). Two ECM conditions were used during cultures: Matrigel versus ECM obtained from pig brain. Subsequently, the addition of human ECM will be tested. Finally, organoids are being characterized using different techniques such as immunofluorescence, RT-PCR and expression arrays.Results: We have found molecular differences among brain organoids obtained following the different protocols, as those obtained following Rosebrock’s protocol express fewer endodermal and mesodermal markers than those obtained with Lancaster's protocol. As well, we have identified different features between those organoids matured with ECM and those matured only with Matrigel, the former being larger than the latter. Further studies will discern whether there are other relevant differences between the models

Procedimiento Estimación Incertidumbre En Un Sistema De Medida Caso Magnetómetro Hall

En este trabajo se presenta un método para estimar el valor de incertidumbre asociada a la medida por un sistema de medición electrónico, como componente metrológico en el proceso de diseño y construcción de un sistema de medida genérico. En este caso especifico, para un magnetómetro Hall de precisión. El método usado es por comparación con un equipo patrón. También se determina el error en la medida aportado por los diferentes componentes del sistema de acondicionamiento en el sistema de medida. El procedimiento plantea un valor para la incertidumbre en la medida de campo magnético de 0.8 % y un error de 0.2 %. El error es aportado por los diferentes componentes del sistema de instrumentación

Procedimiento Estimación Incertidumbre En Un Sistema De Medida Caso Magnetómetro Hall

En este trabajo se presenta un método para estimar el valor de incertidumbre asociada a la medida por un sistema de medición electrónico, como componente metrológico en el proceso de diseño y construcción de un sistema de medida genérico. En este caso especifico, para un magnetómetro Hall de precisión. El método usado es por comparación con un equipo patrón. También se determina el error en la medida aportado por los diferentes componentes del sistema de acondicionamiento en el sistema de medida. El procedimiento plantea un valor para la incertidumbre en la medida de campo magnético de 0.8 % y un error de 0.2 %. El error es aportado por los diferentes componentes del sistema de instrumentación

Participation of gai-adenylate cyclase and ERK1/2 in mas receptor signaling pathways

The MasR receptor (MasR) is an orphan G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang-(1-7) protective axis of renin-angiotensin system. This receptor has been suggested to participate in several physiological processes including cardio- and reno-protection and regulation of the central nervous system function. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, these are still poorly understood. Accordingly, in the current study we aimed to characterize the signaling pathways triggered by the MasR. To do that, we measured cAMP and Ca2+ levels in both naïve and MasR transfected cells in basal conditions and upon incubation with putative MasR ligands. Besides, we evaluated activation of ERK1/2 by Ang-(1-7) in MasR transfected cells. Results indicated the existence of a high degree of MasR constitutive activity toward cAMP modulation. This effect was not mediated by the PDZ-binding motif of the MasR but by receptor coupling to Gai-adenylyl cyclase signaling pathway. Incubation of MasR transfected cells with Ang-(1-7) or the synthetic ligand AVE 0991 amplified MasR negative modulation of cAMP levels. On the other hand, we provided evidence for lack of MasR-associated modulation of Ca2+ levels by Ang-(1-7). Finally, it was determined that the MasR attenuated Ang-(1-7)-induced ERK1/2 phosphorylation mediated by AT1R. We provided further characterization of MasR signaling mechanisms regarding its constitutive activity and response to putative ligands. This information could prove useful to better describe MasR physiological role and development of therapeutic agents that could modulate its action.Fil: Burghi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Echeverría, Emiliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Sosa, Máximo Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Quiroga, Diego Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Muñoz, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Monczor, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Fernandez, Natalia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Dominici, Fernando Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Immune Correlates of Natural HIV Elite Control and Simultaneous HCV Clearance—Supercontrollers

HIV-elite controllers are a minority group of HIV-infected patients with the ability to maintain undetectable HIV viremia for long time periods without antiretroviral treatment. A small group of HIV-controllers are also able to spontaneously clear the hepatitis C virus (HCV) whom we can refer to as “supercontrollers.” There are no studies that explore immune correlates looking for the mechanisms implicated in this extraordinary phenomenon. Herein, we have analyzed HCV- and HIV-specific T-cell responses, as well as T, dendritic and NK cell phenotypes. The higher HCV-specific CD4 T-cell polyfunctionality, together with a low activation and exhaustion T-cell phenotype was found in supercontrollers. In addition, the frequency of CD8 CD161high T-cells was related with HIV- and HCV-specific T-cells polyfunctionality. Interesting features regarding NK and plasmacytoid dendritic cells (pDCs) were found. The study of the supercontroller's immune response, subjects that spontaneously controls both chronic viral infections, could provide further insights into virus-specific responses needed to develop immunotherapeutic strategies in the setting of HIV cure or HCV vaccination

Sostenibilidad en la Ingeniería Civil. Una experiencia piloto de formación profesional y aprendizaje cooperativo

A Pilot Experience on Cooperative Learning was developed with vocational training students of Advanced Level in Building Construction and Civil Engineering of the Atenea Secondary School (Ciudad Real). The aim was to improve their professional skills in the field of sustainability and, in particular, wastewater treatment by planning, designing, projecting and building artificial wetlands; this a innovative and distinctive competence that will facilitate their employment in an environment with demand for skilled technicians in water purification systems that are more sustainable in both environmental, energy and economical terms. For this purpose, a transdisciplinary team of university professors was formed, who are specialized on building and civil engineering, environmental technology, ecology, ecological engineering, hydraulics, hydrology, geotechnics, geology, geomorphology, geography, territorial planning, teaching and pedagogy. The methodology used was Project Based Learning (PBL), that has been used for some of the team members since 1999 in the Faculty of Civil Engineering of the Universidad de Castilla-La Mancha (UCLM). Students were asked to propose, design and project an artificial wetland to treat wastewater from a rural home, museum or restaurant that they had projected earlier to rehabilitate the ruins of a 19th century water-energy-industrial foundry known as Martinete de Los Pozuelos de Calatrava (Ciudad Real). The major milestones of the pilot experience were: i) Pre-assessment of students knowledge, ii) Lectures to provide new theoretical concepts essential to develop foreseen specific, iii) Independent student work supervised by teachers from closed scripts and supply of teaching materials and literature, iv) Classroom and field workshops, v) Continuous assessment of individual work in class (interest, participation, success in the resolution of the issues raised by the teacher), final evaluation in group through oral presentation and written report, and issuing diplomas for further recognition of free credits, vi) Evaluation of the pilot experience was done by surveying students about methodology, content and development of classes, results obtained and other comments that they wished to include, vii) Dissemination by opening an account in the facebook social network, writing a press release for publication in local media, and professional editing a CD with graphic and teaching materials. The most remarkable result of the pilot is that PBL methodology is a revolution for vocational training students, used to deal with tools and practicalities commonly based on closed standards and protocols; instead, cooperative learning requires facing real limitations, such as a relative lack of information, developing the project with this uncertainty, and making decisions on the level of complexity of the solutions to adopt. This has resulted in an overall very positive experience for the students (over 95%), who were especially pleased with the results (100%) and the content and development of educational activities (more than 95%)

ENE-COVID nationwide serosurvey served to characterize asymptomatic infections and to develop a symptom-based risk score to predict COVID-19

Objectives: To characterize asymptomatic SARS-CoV-2 infections and develop a symptom-based risk score useful in primary healthcare. Study design and setting: Sixty-one thousand ninty-two community-dwelling participants in a nationwide population-based serosurvey completed a questionnaire on COVID-19 symptoms and received an immunoassay for SARS-CoV-2 IgG antibodies between April 27 and June 22, 2020. Standardized prevalence ratios for asymptomatic infection were estimated across participant characteristics. We constructed a symptom-based risk score and evaluated its ability to predict SARS-CoV-2 infection. Results: Of all, 28.7% of infections were asymptomatic (95% CI 26.1-31.4%). Standardized asymptomatic prevalence ratios were 1.19 (1.02-1.40) for men vs. women, 1.82 (1.33-2.50) and 1.45 (0.96-2.18) for individuals <20 and ≥80 years vs. those aged 40-59, 1.27 (1.03-1.55) for smokers vs. nonsmokers, and 1.91 (1.59-2.29) for individuals without vs. with case contact. In symptomatic population, a symptom-based score (weights: severe tiredness = 1; absence of sore throat = 1; fever = 2; anosmia/ageusia = 5) reached standardized seroprevalence ratio of 8.71 (7.37-10.3), discrimination index of 0.79 (0.77-0.81), and sensitivity and specificity of 71.4% (68.1-74.4%) and 74.2% (73.1-75.2%) for a score ≥3. Conclusion: The presence of anosmia/ageusia, fever with severe tiredness, or fever without sore throat should serve to suspect COVID-19 in areas with active viral circulation. The proportion of asymptomatics in children and adolescents challenges infection control.The ENE-COVID study was supported by the Spanish Ministry of Health, the Institute of Health Carlos III, and the Spanish National Health System. The funders were in- volved in the study logistics, but they had no role in study design or in the collection, analysis, interpretation of data, or the decision to submit the article for publicationS

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis

Evolution of antibodies against SARS-CoV-2 over seven months: experience of the Nationwide Seroprevalence ENE-COVID Study in Spain [preprint]

Objectives To analyse temporal trends in SARS-CoV-2 anti-nucleocapsid IgG throughout the four rounds of the nationwide seroepidemiologic study ENE-COVID (April-November 2020), and to compare the fourth-round results of two immunoassays detecting antibodies against nucleocapsid and to S protein receptor-binding domain (RBD). Methods A chemiluminescent microparticle immunoassay (CMIA) was offered to all participants in the first three rounds (Abbott; anti-nucleocapsid IgG). In the fourth round we offered this test and a chemiluminescence immunoassay (CLIA) (Beckman; anti-RBD IgG) to i) a randomly selected sub-cohort, ii) participants who were IgG-positive in any of the three first rounds; and iii) participants who were IgG-positive in the fourth round by point-of-care immunochromatography. Results Immunoassays involving 10,153 participants (82.2% of people invited to donate samples) were performed in the fourth round. A total of 2595 participants (35.1% of participants with immunoassay results in the four rounds) were positive for anti-nucleocapsid IgG in at least one round. Anti-nucleocapsid IgG became undetectable in 43.3% of participants with positive first-round results. Pneumonia was more frequent in participants with anti-nucleocapsid IgG in all four rounds (11.2%) than those in which IgG became undetectable (2.4%). In fourth round, anti-nucleocapsid and anti-RBD IgG were detected in 5.5% and 5.4% participants of the randomly selected sub-cohort, and in 26.6% and 25.9% participants with at least one previous positive result, respectively. Agreement between techniques was 90.3% (kappa: 0.72). Conclusions The response of IgG to SARS-CoV-2 is heterogeneous and conditioned by infection severity. A substantial proportion of the SARS-CoV-2 infected population may have negative serologic results in the post-infection months.N

Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (Ka: 1560 ± 20 M-1). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested lycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design