Inhibition of Crif1 protects fatty acid-induced POMC neuron-like cell-line damage by increasing CPT-1 function.

Hypothalamic proopiomelanocortin (POMC) neurons are sensors of signals that reflect the energy stored in the body. Inducing mild stress in proopiomelanocortin neurons protects them from the damage promoted by the consumption of a high-fat diet, mitigating the development of obesity; however, the cellular mechanisms behind these effects are unknown. Here, we induced mild stress in a proopiomelanocortin neuron cell line by inhibiting Crif1. In proopiomelanocortin neurons exposed to high levels of palmitate, the partial inhibition of Crif1 reverted the defects in mitochondrial respiration and ATP production; this was accompanied by improved mitochondrial fusion/fission cycling. Furthermore, the partial inhibition of Crif1 resulted in increased reactive oxygen species production, increased fatty acid oxidation, and reduced dependency on glucose for mitochondrial respiration. These changes were dependent on the activity of CPT-1. Thus, we identified a CPT-1-dependent metabolic shift toward greater utilization of fatty acids as substrates for respiration as the mechanism behind the protective effect of mild stress against palmitate-induced damage of proopiomelanocortin neurons.NEW & NOTEWORTHY Saturated fats can damage hypothalamic neurons resulting in positive energy balance, and this is mitigated by mild cellular stress; however, the mechanisms behind this protective effect are unknown. Using a proopiomelanocortin cell line, we show that under exposure to a high concentration of palmitate, the partial inhibition of the mitochondrial protein Crif1 results in protection due to a metabolic shift warranted by the increased expression and activity of the mitochondrial fatty acid transporter CPT-1.

The impact of a story on learning ketone body metabolism.

Biochemistry is a core subject in the cross-disciplinary training on Biotechnology engineering courses. Metabolic pathways teaching has traditionally integrated hands-on laboratory experiences and traditional lectures, which detail a large number of reactions at a molecular level, their enzymes and regulation. The current scenario of Covid-19 outbreak have motivated the development of complementary tools that expand the horizon of metabolism teaching. In this study, we employed a story-based methodology to strengthen the metabolic pathways learning and to measure students' perception. Specifically, a peer-reviewed tale describing the ketone body metabolism was used during five semesters as a didactic strategy to teach this biochemical process. A questionnaire assessed the students' understanding and acceptance of the methodology (n = 83). Our findings showed that a high proportion of students (83.13%) were able to relate the story to the topics studied in the classroom (ketogenesis and ketolysis). On the other hand, they were satisfied and suggested that such methodology is effective and fun. In summary, most of the survey responses related to acceptance of story-based strategy ranged from 72% to 97%. Collectively, these results indicated that the story is appropriate to decomplex pathways, becoming a simple tool for driving motivation, learning and engagement of students. The narrative represents a bridge to connect the intriguing series of chemical reactions involved in the anabolism and degradation of 3-hydroxybutyrate (3-OHB), acetoacetate, and acetone with previously learned knowledge, emotions, and key concepts. In conclusion, the tale was useful to decode ketone body-related pathways and making metabolism learning more interesting and easier.

Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far beyond the originally described gene editing. The CRISPR activation (CRISPRa) system combines the catalytically inactive Cas9 (dCas9) protein with distinct transcription modules to induce endogenous gene expression. SunTag-p65-HSF1 (SPH) is a recently developed CRISPRa technology that combines components of synergistic activation mediators (SAMs) with the SunTag activators. This system allows the overexpression of single or multiple genes by designing a customized single-guide RNA (sgRNA). In this study, a previously developed SPH mouse was used to generate a conditional mouse expressing SPH in adipocytes (adiponectin Cre lineage), named AdipoSPH. To induce a white-to-beige fat (browning) phenotype, an adeno-associated virus (AAV) carrying sgRNA targeting the endogenous Prdm16 gene (a well-established transcription factor related to brown and beige fat development) was injected into the inguinal white adipose tissue (iWAT). This mouse model induced the expression of endogenous Prdm16 and activated the thermogenic gene program. Moreover, in vitro SPH-induced Prdm16 overexpression enhanced the oxygen consumption of beige adipocytes, phenocopying the results of a previous Prdm16 transgenic mouse model. Thus, this protocol describes a versatile, cost-effective, and time-effective mouse model for investigating adipose tissue biology.

An experimental protocol for molecular biology lab at an Amazonian University.

Laboratory-based practical classes are an essential component in teaching molecular biology for undergraduate students. Universidad Regional Amazonica Ikiam is a higher education institution located in the Ecuadorian Amazon rainforest, a high biodiversity place, including amphibians. Based on this, we have established a practical molecular biology program with eight sessions that contextualize the biodiverse surroundings of the University. This program stimulates synchronization of information between theory and practice and improves research skills. During these sessions, students are motivated to identify and characterize antimicrobial peptides from Ecuadorian frog skin secretions, using molecular biology techniques and biochemistry and microbiology knowledge. This practical course was held twice with a total of 56 students from the fifth semester of the biotechnology engineering. The evaluation of the practical program was carried out through a questionnaire applied to students using the Likert scale. Overall, this form of teaching had high receptivity and presented benefits for student learning. Interestingly, 80% of respondents strongly agreed that this course provided tools and knowledge for the development of their undergraduate dissertation. Therefore, practical courses tailored to the student's context can stimulate student learning and interest. Additionally, this experimental methodology is interdisciplinary and can be applied to other research fields and subjects.