In vivo imaging of calcium dynamics in zebrafish hepatocytes.

BACKGROUND AND AIMS: Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. APPROACH AND RESULTS: We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. CONCLUSIONS: We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.

Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line that monitors pax2a expression in the thyrocytes. A population of pax2a-low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a-high and pax2a-low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles.

BACKGROUND AIMS: This article describes the development of a small-scale model for Ficoll-based cell separation as part of process development of an advanced therapy medicinal product and its qualification. Because of the complexity of biological products, their manufacturing process as well as characterization and control needs to be accurately understood. Likewise, scale-down models serve as an indispensable tool for process development, characterization, optimization and validation. This scale-down model represents a cell processor device widely used in advance therapies. This approach is inteded to optimise resources and to focus its use on process characterisation studies under the paradigm of the Quality by design. A scale-down model should reflect the large manufacturing scale. Consequently, this simplified system should offer a high degree of control over the process parameters to depict a robust model, even considering the process limitations. For this reason, a model should be developed and qualified for the intended purpose. METHODS: Process operating parameters were studied, and their resulting performance at full scale was used as a baseline to guide scale-down model development. Once the model was established, comparability runs were performed by establishing standard operating conditions with bone marrow samples. These analyses showed consistency between the bench and the large scale. Additionally, statistical analyses were employed to demonstrate equivalence. RESULTS: The process performance indicators and assessed quality attributes were equivalent and fell into the acceptance ranges defined for the large-scale process. CONCLUSIONS: This scale-down model is suitable for use in process characterization studies.

Starvation resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that while surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Further, down-regulation of FATP2 in drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionary conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

Starvation-resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that although surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Furthermore, down-regulation of FATP2 in Drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionarily conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

Articular cartilage delamination at eight years following cellular-based repair procedures: a case reports.

This report describes two cases of late cartilage delamination in two young adults after two different autologous cell-based techniques for cartilage restoration: 1. Matrix-assisted autologous chondrocyte implantation (MACI) and 2. Hyaluronic acid-bone marrow aspirate concentrate (HA-BMAC). Both cases demonstrate that even in patients who do not present with any ongoing symptoms after primary surgery, a cellular-based graft's subsequent delamination can occur later. It is possible that regardless of the technique used or the time passed since the surgery, a graft failure may occur at some level, causing delamination of a previously asymptomatic cartilage restoration graft and a traumatic event with long-term follow-up. Surgeons must be alert to this injury and describe histologic findings to determine where failure occurs.

Abnormal cannabidiol ameliorates inflammation preserving pancreatic beta cells in mouse models of experimental type 1 diabetes and beta cell damage.

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies, including autoimmune diseases. However, its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week, following STZ challenge, and euthanized 1 week later. Blood, pancreas, pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice, treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly, Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice, Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-κB and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether, Abn-CBD reduces circulating and intra-islet inflammation, preserving islets, thus delaying the progression of insulitis. Hence, Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D.

Platelets and Adipose Stroma Combined for the Treatment of the Arthritic Knee.

Knee osteoarthritis (OA) leads to significant pain and disability, prompting new cell-based injections to lessen the symptoms. Biological therapies such as autologous microfragmented adipose tissue (AMAT) and a stromal vascular fraction (SVF) are a common source for harvesting mesenchymal and progenitor cells. Platelet-rich plasma (PRP) is associated with cytokines and growth factors. Recent studies have reported good clinical outcomes with AMAT, SVF, and PRP in knee osteoarthritis treatment. However, the preparation, processing, and application technique are vital to achieving satisfactory results. Many studies have examined outcomes after AMAT, SVF, or PRP injection, with encouraging results. Still, there is a lack of studies describing a technique that combines both methods, the timing, and the amount of SVF or PRP injected. This technical note's objective was to describe a standardized new technique composed of platelet and adipose stroma to treat knee osteoarthritis (OA) and the processing method.

The Atypical Cannabinoid Abn-CBD Reduces Inflammation and Protects Liver, Pancreas, and Adipose Tissue in a Mouse Model of Prediabetes and Non-alcoholic Fatty Liver Disease.

Background and Aims: The synthetic atypical cannabinoid Abn-CBD, a cannabidiol (CBD) derivative, has been recently shown to modulate the immune system in different organs, but its impact in obesity-related meta-inflammation remains unstudied. We investigated the effects of Abn-CBD on metabolic and inflammatory parameters utilizing a diet-induced obese (DIO) mouse model of prediabetes and non-alcoholic fatty liver disease (NAFLD). Materials and Methods: Ten-week-old C57Bl/6J mice were fed a high-fat diet for 15 weeks, following a 2-week treatment of daily intraperitoneal injections with Abn-CBD or vehicle. At week 15 mice were obese, prediabetic and developed NAFLD. Body weight and glucose homeostasis were monitored. Mice were euthanized and blood, liver, adipose tissue and pancreas were collected and processed for metabolic and inflammatory analysis. Results: Body weight and triglycerides profiles in blood and liver were comparable between vehicle- and Abn-CBD-treated DIO mice. However, treatment with Abn-CBD reduced hyperinsulinemia and markers of systemic low-grade inflammation in plasma and fat, also promoting white adipose tissue browning. Pancreatic islets from Abn-CBD-treated mice showed lower apoptosis, inflammation and oxidative stress than vehicle-treated DIO mice, and beta cell proliferation was induced. Furthermore, Abn-CBD lowered hepatic fibrosis, inflammation and macrophage infiltration in the liver when compared to vehicle-treated DIO mice. Importantly, the balance between hepatocyte proliferation and apoptosis was improved in Abn-CBD-treated compared to vehicle-treated DIO mice. Conclusions: These results suggest that Abn-CBD exerts beneficial immunomodulatory actions in the liver, pancreas and adipose tissue of DIO prediabetic mice with NAFLD, thus protecting tissues. Therefore, Abn-CBD and related compounds could represent novel pharmacological strategies for managing obesity-related metabolic disorders.

School Achievement and Performance in Chilean High Schools: The Mediating Role of Subjective Wellbeing in School-Related Evaluations.

School achievement gaps and school failure are problematic issues in Latin America, and are mainly explained by the socio-economic status (SES) of the students. What schools can do to improve school achievement and reduce school failure is a critical issue, both for school management and teacher training. In this study, we present the association of individual and school-related socio-emotional variables with school achievement and performance, controlling for the effects of SES. A probabilistic sample of 4,964 students, drawn from 191 schools enrolled in year 10 in urban areas of Chile, answered questionnaires assessing subjective wellbeing, social wellbeing in school, school climate, school social wellbeing and students' perceptions of teachers' wellbeing. Using structural equation modeling, and controlling for SES, we modeled subjective wellbeing as a mediator of the relationship between school-related variables, such as school climate and perception of teacher's wellbeing, and (a) school achievement, and (b) school performance. School achievement was computed as a product of (a) the probability of passing the school year, and (b) the percentage of yearly attendance at school. Data on school achievement was drawn from administrative registries from the Chilean Ministry of Education. School performance was computed as the estimated grade point average (GPA) at the end of the school year, based on the students' previous 5-year GPAs, and was also obtained through administrative data of the last 5 years. Findings reveal the mediating role of subjective wellbeing in the relationship between school-related evaluations (students' social wellbeing at school, their perception of teachers' wellbeing and school climate) and school achievement. For school achievement, two variables were mediated (students' social wellbeing at school and school climate). However, for school performance, no significant mediations were found. We conclude that, on the one hand, after controlling for SES, students' individual subjective wellbeing is associated with their achievement and performance in school. We discuss the importance of improving school experiences that may protect and promote students' subjective experience and school achievement and performance, and reduce the probability of school failure and dropout.

Management of enzyme diversity in high-performance cellulolytic cocktails.

BACKGROUND: Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. RESULTS: One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. CONCLUSIONS: These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Relación entre segregación y convivencia escolar en escuela públicas chilenas / Relationship between segregation and school climate in Chilean schools with high scores PISA 2009

La educación chilena es altamente segregadora, lo que dificulta la construcción de sociedades cohesionadas y democráticas. La investigación corresponde a un diseño mixto, secuencial y bietápico que indagó la relación entre segregación (selección y nivel socioeconómico) y calidad de la convivencia escolar, tomando como muestra las 200 escuelas PISA 2009. Los resultados evidencian peores ambientes escolares en las escuelas municipales, de niveles socioeconómicos bajos y no selectivos, donde los estudiantes se sienten estigmatizados y discriminados por variables académicas, familiares y políticas. Se discute la necesidad de revisar la política educativa que incentiva la competencia entre escuelas por rendimiento académico y formar en educación cívica para avanzar hacia escuelas más integradas y con ello a sociedades más justas y democráticas.

Chilean education is highly segregated, making it difficult to construct cohesive and democratic societies. Through a mixed-method sequential design we explored the relationship between segregation (selection and socioeconomic level) and quality of school climate, taking as example the 200 Chilean schools who participated in the PISA 2009 test. The results evidence worst school environments in public, non-selective and low socioeconomic status schools, where students feel stigmatized and discriminated by academic, social, family and political variables. We discuss the need to revise the education policy that encourages competition among schools for academic performance and training in civics to move towards more integrated schools and more democratic societies.

Escala de Clima Escolar: adaptación al español y validación en estudiantes chilenos / School Climate Scale: Spanish Adaptation and Validation in Chilean Students

El clima escolar resulta de creciente interés para investigadores e instituciones educativas. Las estrategias de mejoramiento escolar basadas en evidencias han incluido el clima escolar para apoyar a los integrantes de comunidades educativas en la creación de escuelas seguras y comprometidas con el aprendizaje. Es necesario contar con instrumentos validados para la población latinoamericana, que permitan la investigación comparada. Presentamos los resultados de la validación de la Escala de Clima Escolar. El instrumento mide la percepción de estudiantes sobre la aplicación de normas, el apoyo de adultos y la participación. Se tradujo al español y se realizó un análisis psicométrico. La muestra fue de 4688 estudiantes chilenos, de 4°, 6° y 8° básico. Los resultados evidencian un alto nivel de consisten interna (a = 0.89) y de varianza explicada (54%). El modelo de tres factores mostró un buen ajuste, así como uno de cuatro factores, que separa las normas generales de las normas contra la violencia sexual.

School climate has received increased attention use by researchers and educational institutions. Evidence-based school improvement processes have included school climate as a means of helping create schools that are safe and committed with learning. It is necessary to count with valid measures of school climate for Latin American countries, which may allow for comparative research. We present the results of the validation of the School Climate Scale. The instrument measures students' perceptions of norms, support from adults in the school, and participation. The scale was translated into Spanish and its psychometric properties were studied on a sample of 4,688 Chilean students from grades fourth, sixth, and eight. Findings show a high level on internal consistency (α = 0.89) and explained variance (54%). Structural equation models fit the data well, both for the three-factor solution as well a four-factor structure that separates general school norms from norms against sexual violence. The scale can facilitate data- driven decisions and may be incorporated into evidenced-based school improvement processes.

El papel mediador de la capacitación docente en el manejo de la violencia escolar sobre el bienestar social de profesores / The mediating role of teacher training in the management of school violence on social welfare of teachers

La violencia escolar afecta negativamente, y el bienestar social escolar positivamente, la salud mental de los estudiantes y de sus profesores. En este estudio se analizó el efecto mediador de la necesidad de contar con capacitación en violencia escolar y de la magnitud de violencia escolar, en 397 profesores de escuelas básicas, sobre la asociación entre la frecuencia percibida de maltrato entre estudiantes y la coherencia social con que observan su contexto escolar. El modelo de mediación resultó significativo y mejoró el poder predictivo de la frecuencia de conductas de maltrato entre estudiantes sobre la coherencia social de la escuela. Sin embargo, es solo la percepción que los profesores tienen sobre su necesidad de capacitación en violencia escolar la variable que actúa como mediadora. Se discuten estos hallazgos en relación a la necesidad de fortalecer y mejorar los programas de capacitación y formación inicial y continua de los docentes.

Students' and teachers' mental health is negatively affected by school violence, and positively affected by teachers' social wellbeing in school. We analyzed the mediating effect of teachers' perceived need to receive training in school violence prevention, as well as teachers' perceived magnitude of violence in their schools, on the negative association between the frequency of student maltreatment and teachers' perception of social coherence in their school. 397 elementary school teachers participated in this study. The mediating model theorized proved significant and improved the predictive power of the frequency of student maltreatment on teachers' social coherence in school. However, only teachers' perceptions of the need to receive teacher training on school violence had a mediating effect. Findings are discussed in terms of the need to strengthen and improve teacher training programs in school violence and school wellbeing, both for in-service as well as for teachers in formation.