A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting

GPR50 Interacts with TIP60 to Modulate Glucocorticoid Receptor Signalling

GPR50 is an orphan G-protein coupled receptor most closely related to the melatonin receptors. The physiological function of GPR50 remains unclear, although our previous studies implicate the receptor in energy homeostasis. Here, we reveal a role for GPR50 as a signalling partner and modulator of the transcriptional co-activator TIP60. This interaction was identified in a yeast-two-hybrid screen, and confirmed by co-immunoprecipitation and co-localisation of TIP60 and GPR50 in HEK293 cells. Co-expression with TIP60 increased perinuclear localisation of full length GPR50, and resulted in nuclear translocation of the cytoplasmic tail of the receptor, suggesting a functional interaction of the two proteins. We further demonstrate that GPR50 can enhance TIP60-coactiavtion of glucocorticoid receptor (GR) signalling. In line with in vitro results, repression of pituitary Pomc expression, and induction of gluconeogenic genes in liver in response to the GR agonist, dexamethasone was attenuated in Gpr50−/− mice. These results identify a novel role for GPR50 in glucocorticoid receptor signalling through interaction with TIP60

Exploring the effects of near-peer teaching in robotics education: The role of STEM attitudes

Due to technological advancements, robotics is findings its way into the classroom. However, workload for teachers is high, and teachers sometimes lack the knowledge to implement robotics education. A key factor of robotics education is peer learning, and having students (near-)peers teach them robotics could diminish workload. Therefore, this study implemented near-peer teaching in robotics education. 4 K10-11 secondary school students were teachers to 83 K5-6 primary school students. The intervention included 4 3-hour robotics lessons in Dutch schools. Primary school students completed a pre- and post-intervention questionnaire on their STEM-attitudes and near-peer teaching experience, and a report on their learning outcomes. Interaction with near-peer teachers was observed. After the lessons, a paired-samples t-test showed that students had a more positive attitude towards engineering and technology. Students also reported a positive near-peer teaching experience. Conventional content analysis showed that students experienced a gain in programming and robotics skill after the lessons, and increased conceptual understanding of robotics. The role the near peer teachers most frequently fulfilled was formative assessor. Near-peer teachers could successfully fulfil a role as an engaging information provider. This study shows that near-peer teachers can effectively teach robotics, diminishing workload for teachers. Furthermore, near-peer robotics lessons could lead to increased STEM-attitudes

Umsetzung anästhesiologischer Fast-Track-Maßnahmen bei kolorektalen Resektionen

Purpose!#!The fast-track (FT) concept is a multimodal, interdisciplinary approach to perioperative patient care intended to reduce postoperative complications. Despite good evidence implementation seems to need improvement, whereby almost all studies focused on the implementation of surgical modules regardless of the interdisciplinary aspect. Adherence to the anesthesiological measures (prehabilitation, premedication, volume and temperature management, pain therapy), on the other hand, has been insufficiently studied. To assess the status quo a survey on the implementation of anesthesiological FT measures was conducted among members of the German Society of Anesthesiology and Intensive Care Medicine (DGAI) to analyze where potential for improvement exists.!##!Methods!#!Using the SurveyMonkey® online survey tool, 28 questions regarding perioperative anesthesiological care of colorectal surgery patients were sent to DGAI members in order to analyze adherence to FT measures.!##!Results!#!While some of the FT measures (temperature management, PONV prophylaxis) are already routinely used, there is a divergence between current recommendations and clinical implementation for other components. In addition to premedication, interdisciplinary measures (prehabilitation) and measures that affect multiple interfaces (operating theatre, recovery room, ward), such as volume management or perioperative pain management, are particularly affected.!##!Conclusion!#!The anesthesiological recommendations of the FT concept are only partially implemented in Germany. This particularly affects the interdisciplinary components as well as measures at the operating theatre, recovery room and ward interfaces. The establishment of an interdisciplinary FT team and interdisciplinary development of SOPs can optimize adherence, which in turn improves the short-term and long-term outcome of patients

Melanocortin-4 Receptor Gene Mutations in a Dutch Cohort of Obese Children

Epidemiology in Pediatrics and Child Healt

Transcutaneous vagal nerve simulation to reduce a systemic inflammatory response syndrome and the associated intestinal failure: study protocol of a prospective, two-armed, sham-controlled, double-blinded trial in healthy subjects (the NeuroSIRS-Study)

Purpose!#!Surgery initiates pro-inflammatory mediator cascades leading to a variably pronounced sterile inflammation (SIRS). SIRS is associated with intestinal paralysis and breakdown of intestinal barrier and might result in abdominal sepsis. Technological progress led to the development of a neurostimulator for transcutaneous auricular vagal nerve stimulation (taVNS), which is associated with a decline in inflammatory parameters and peristalsis improvement in rodents and healthy subjects via activation of the cholinergic anti-inflammatory pathway. Therefore, taVNS might be a strategy for SIRS prophylaxis.!##!Methods!#!The NeuroSIRS-Study is a prospective, randomized two-armed, sham-controlled, double-blind clinical trial. The study is registered at DRKS00016892 (09.07.2020). A controlled endotoxemia is used as a SIRS-mimicking model. 2 ng/kg bodyweight lipopolysaccharide (LPS) will be administered after taVNS or sham stimulation. The primary objective is a reduction of clinical symptoms of SIRS after taVNS compared to sham stimulation. Effects of taVNS on release of inflammatory cytokines, intestinal function, and vital parameters will be analyzed.!##!Discussion!#!TaVNS is well-tolerated, with little to no side effects. Despite not fully mimicking postoperative inflammation, LPS challenge is the most used experimental tool to imitate SIRS and offers standardization and reproducibility. The restriction to healthy male volunteers exerts a certain bias limiting generalizability to the surgical population. Still, this pilot study aims to give first insights into taVNS as a prophylactic treatment in postoperative inflammation to pave the way for further clinical trials in patients at risk for SIRS. This would have major implications for future therapeutic approaches

Suppressor of cytokine signaling 3 knockdown in the mediobasal hypothalamus: counterintuitive effects on energy balance

An increase in brain suppressor of cytokine signaling 3 (SOCS3) has been implicated in the development of both leptin and insulin resistance. Socs3 mRNA is localized throughout the brain, and it remains unclear which brain areas are involved in the effect of SOCS3 levels on energy balance. We investigated the role of SOCS3 expressed in the mediobasal hypothalamus (MBH) in the development of diet-induced obesity in adult rats. Socs3 mRNA was down-regulated by local injection of adeno-associated viral vectors expressing a short hairpin directed against Socs3, after which we determined the response to high-fat high-sucrose choice diet. In contrast to neuronal Socs3 knockout mice, rats with SOCS3 knockdown limited to the MBH showed increased body weight gain, larger amounts of white adipose tissue, and higher leptin concentrations at the end of the experiment. These effects were partly due to the decrease in locomotor activity, as 24 h food intake was comparable with controls. In addition, rats with Socs3 knockdown in the MBH showed alterations in their meal patterns: average meal size in the light period was increased and was accompanied by a compensatory decrease in meal frequency in the dark phase. In addition, neuropeptide Y (Npy) mRNA levels were significantly increased in the arcuate nucleus of Socs3 knockdown rats. Since leptin is known to stimulate Npy transcription in the absence of Socs3, these data suggest that knockdown of Socs3 mRNA limited to the MBH increases Npy mRNA levels, which subsequently decreases locomotor activity and alters feeding pattern