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

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

The development and validation of a handicap questionnaire for children with a chronic illness

Introduction: This paper describes the development and initial psychometric evaluation of the Handicap Scale for Children (HSC). This questionnaire is based on the London Handicap Scale (LHS), a valid and reliable utility instrument for measuring social participation in adults. Methods: A multidisciplinary research group was involved in developing the HSC. The questionnaire was tested in 114 children with a chronic disease and 239 healthy children in the 8-18 age range. Relating the Health Utility Index Mark 3 (HUI3) attributes to corresponding HSC scores tested the assumption that a negative health status would lead to participation problems. Results: Questionnaire development resulted in a five-dimension questionnaire: mobility, physical independence, daily activities, social integration and orientation. Each dimension included one item with a six-point response scale. A higher score indicates greater handicap. Feasibility testing with 10 children showed that none of the children experienced difficulties in filling in the questionnaire. Conceptual validity, measured by correlations between the dimensions of the HSC and HUI3, was satisfactory. As expected, moderate correlation coefficients between predefined pairs of HUI and HSC attributes were found; other correlation coefficients were low. Criterion validity was also satisfactory, as shown by large differences between the healthy and the chronically ill group and by several criteria within the chronically ill group. Conclusion: Based on this initial evaluation, the questionnaire seems feasible and valid for use with children in the age range 8-18 years. © 2005 Edward Arnold (Publishers) Ltd

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization.

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses

Associations entre le retard de croissance et l'émaciation à l'âge de 12 mois et les retards de développement dans une cohorte d'enfants cambodgiens

International audienceAbstract Worldwide, over 250 million children under 5 years do not reach their developmental potential due to several causes, including malnutrition. In Cambodia, the prevalence of stunting and wasting among children remains high. This prospective cohort study aimed to assess acquisition of motor and cognitive developmental milestones in early childhood and their associations with stunting and wasting. Children aged from 0 to 24 months were recruited from three provinces in Cambodia and followed up to seven times from March 2016 to June 2019, until their 5 years. Data collection included anthropometry and developmental milestones. Seven motor and seven cognitive milestones were evaluated using the Cambodian Development Milestone Assessment Tool. Associations were assessed with parametric survival models. Hazard ratios (HR) below 1 stood for lower probabilities for achieving developmental milestones. Data were available for 7394 children. At 12 months, the prevalence of stunting and wasting were 23.7% and 9.6% respectively. Both were consistently associated with delays in most motor and cognitive milestones. Stunting was strongly associated with delays in gross motor milestones (HR < 0.85; p < 0.001). Wasting was more strongly associated with delays in fine motor development and most cognitive milestones (HR < 0.75; p < 0.001). Promoting nutritional programs in the first 1000 days to prevent malnutrition is essential to further the optimal growth and motor and cognitive development of Cambodian children

The associations between stunting and wasting at 12 months of age and developmental milestones delays in a cohort of Cambodian children

Worldwide, over 250 million children under 5 years do not reach their developmental potential due to several causes, including malnutrition. In Cambodia, the prevalence of stunting and wasting among children remains high. This prospective cohort study aimed to assess acquisition of motor and cognitive developmental milestones in early childhood and their associations with stunting and wasting. Children aged from 0 to 24 months were recruited from three provinces in Cambodia and followed up to seven times from March 2016 to June 2019, until their 5 years. Data collection included anthropometry and developmental milestones. Seven motor and seven cognitive milestones were evaluated using the Cambodian Development Milestone Assessment Tool. Associations were assessed with parametric survival models. Hazard ratios (HR) below 1 stood for lower probabilities for achieving developmental milestones. Data were available for 7394 children. At 12 months, the prevalence of stunting and wasting were 23.7% and 9.6% respectively. Both were consistently associated with delays in most motor and cognitive milestones. Stunting was strongly associated with delays in gross motor milestones (HR < 0.85; p < 0.001). Wasting was more strongly associated with delays in fine motor development and most cognitive milestones (HR < 0.75; p < 0.001). Promoting nutritional programs in the first 1000 days to prevent malnutrition is essential to further the optimal growth and motor and cognitive development of Cambodian children

The Multiple Endocrine Neoplasia Type 1 (MEN1) Tumor Suppressor Regulates Peroxisome Proliferator-Activated Receptor γ-Dependent Adipocyte Differentiation▿

Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several MEN1-related tumor types, we investigated regulation of PPARγ activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARγ-expressing mouse embryonic fibroblasts. Menin augments PPARγ target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARγ in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARγ. We propose that menin is an important factor in PPARγ-mediated adipogenesis and that loss of PPARγ function may contribute to lipoma development in MEN1 patients