Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate–protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced ‘browning’ of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a ‘replace me’ signalling function that regulates thermogenic fat and counteracts obesity.
Substance P and hemokinin‐1 were predominantly examined by immunoassays with their limitation to differentiate appropriately between both peptides. The use of liquid chromatography coupled with tandem mass spectrometry is a promising, highly selective alternative. Adsorption processes have been identified in preliminary experiments to play a crucial role in the loss of mass spectrometry intensity of both peptides. Therefore, a design of experiments concept was created to minimize nonspecific peptide adsorption. For this purpose, the most critical influencing parameters—(1) the composition of the injection solvent as well as (2) the most suitable container material—were systematically and concordantly investigated. The addition of modifiers, such as formic acid, dimethyl sulfoxide, and organic solvents, to the injection solvent led to a substantial gain of intensity of substance P and hemokinin‐1 compared to the start gradient as an injection solvent. Furthermore, the systematic investigation underlined the high impact of the container material, demonstrating polypropylene as the most favorable material. A conjoint injection solvent optimum was found to determine both peptides simultaneously by the conduction of a sweet‐spot analysis. The experimental design substantially reduced nonspecific peptide adsorption and enabled the simultaneous and selective determination of endogenous substance P and hemokinin‐1 plasma levels.
BackgroundPlasma renin levels were determined in the academia-driven, EU-funded “Labeling of Enalapril from Neonates up to Adolescents” (LENA) project to evaluate its role in pediatric heart failure. Quality-controlled bioanalysis is crucial to ensure reliable data generation. However, a comprehensive bioanalytical quality control (QC) concept to monitor the method performance within an academic environment was lacking.MethodsThus, a QC concept was designed encompassing regulatory guidance, international recommendations and current scientific discussions. The concept included (1) a system-suitability test, (2) verification of single bioanalytical runs by calibration curve performance and evaluation of QCs, (3) assessment of the inter-run accuracy according to Clinical Laboratory Standards Institute (CLSI) guideline, (4) monitoring of reproducibility by pediatric incurred samples, (5) blank-sample analysis and (6) participation in interlaboratory testing.ResultsThe concept was successfully applied to the academic project. About 11% of single runs were identified as invalid and triggered a re-analysis of unknown samples being included in those runs. The usefulness of the customized inter-run monitoring was demonstrated and proved the good accuracy from the first to the last run. All 147 reanalyzed incurred sample pairs complied with regulatory requirements.ConclusionsThe regulatory complied QC concept was customized for the demands of academia-driven pediatric trials and contributed to the reliable quantification of 965 pediatric renin samples.
BackgroundIn Germany 37.1% of dispensed medicinal products were intended to use in self-medication in 2017.1 An investigation showed that 25.2% of children and adolescents used self-medication in Germany.2 Hence, pharmacists’ education needs to include training for competence in consultation.3A modern method to train this competence is the use of OSCEs (Objective Structured Clinical Examinations). The aim of this study was to assess whether the use of OSCEs in pharmacy students to train the consultation performance on self-medication is more effective than a conservative teaching method.MethodsThis randomised controlled investigation was conducted in a pre-post-design with pre-OSCEs before training and post-OSCEs after training in each group. Clinical skills at baseline and after the training were measured. Forty students in their last year of pharmacy studies were randomised into a control and an intervention group. The control group attended a lecture on self-medication and the intervention group had additionally to the lecture one hour of OSCEs for training purpose. An analytical checklist was used for measuring consultation skills and a global rating scale for assessing communication skills.ResultsComplete data was received from 30 students (n=16 intervention group, n=14 control group). Consultation skills improved significantly (analytical checklist: 19.88% ± 10.95% intervention group vs. 9.29% ± 10.89% control group, p< 0.05). However, the communication skills (global rating scale: 20.83% ± 24.33% in the intervention group vs. 11.90% ± 17.12% in the control group, p= 0.380) did not improve significantly during the one-hour training period.ConclusionOSCEs for training purpose are an effective method to convey pharmacy students consultation skills in self-medication. However, communication skills need more training. Based on these results OSCEs on self-medication for the paediatric population should be investigated. This is relevant due to the frequency of self-medication in the paediatric population.ReferencesAbda.de. [Internet]. Berlin: Federal union of German associations of pharmacists. Numbers, data, facts 2016. [Cited January 30, 2019]. Available from: https://www.abda.de/fileadmin/assets/ZDF/ZDF_2018/ABDA_ZDF_2018_Brosch.pdf Du Y, Knopf H. Self-medication among children and adolescents in Germany: results of the National Health Survey for children and adolescents (KiGGS). Br J Clin Pharmacol 2009;68:599–608.Joint Statement by the International Pharmaceutical Federation (FIP) and the World Self Medication Industry (WSMI). [Internet]. Responsible Self-medication. 1998. [Cited January 30, 2019]. Available from: https://www.fip.org/www/uploads/database_file.php?id=241&table_id=.Disclosure(s)Imaneh Farahani, Anna Laven, Samieh Farahani, Maira A. Deters, Martin Feickert, Fabian K. Suessenbach, Holger Schwender and Stephanie Laeer declare that they have no conflict of interest.
BackgroundAs the initiator of the Renin-Angiotensin-Aldosterone-system, renin plays an essential role in the vicious circle of heart failure. Therefore, renin was determined in the investigators driven ‘Labelling of Enalapril from neonates up to adolescents’ (LENA) study to evaluate its role in paediatric heart failure. Due to the often long-lasting periods of recruitment of paediatric subjects, the assay performance has to be guaranteed over the whole recruiting time. Therefore, to ensure the high quality of the determined renin study samples after successful assay validation,1 a multi-step quality approach was used to get reliable results over a period of 30 months.MethodsBased on a multi-step quality approach consisting of calibration standards (CSs), quality controls (QCs) and incurred sample reanalysis (ISR), study samples of unknown renin concentrations were determined. Results within predefined limits of CSs (6 levels) and QCs according to European Medicine Agency (EMA) guidelines were required for evaluating the study samples.2 ISR was performed for randomly selected paediatric samples to evaluate the long-term accuracy of the validated assay.Results133 analytical runs were conducted for renin from February 2016 to August 2018. In 119 (88.8%) valid runs, a total number of 1414 of CCs and 952 of QCs were determined. Thereof 99.9% of CCs and 98.3% of QCs were in the predefined limits according to EMA. 143 incurred sample pairs were reanalysed resulting in 95.8% of samples within EMA guidelines. Using this multi-step quality approach, the reliable determination of 965 LENA paediatric study samples was guaranteed.ConclusionIn addition to the assay validation, the multi-step quality approach ensured the reliability of the determined renin concentrations in the continuous bioanalysis of the paediatric study samples and guaranteed the high quality of the collected data in the LENA study.ReferencesSchaefer J, Burckhardt BB, Tins J, et al. Validated low-volume immunoassay for the reliable determination of direct renin especially valuable for pediatric investigations. J Immunoass Immunochem 2017;38:579–94. doi:10.1080/15321819.2017.1350707Guideline on bioanalytical method validation. European Medicines Agency, London, UK (2011).Disclosure(s)Martin Feickert, Ilja Burdman, Nina Makowski, Moshin Ali, Anke Bartel, and Bjoern B. Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n°602295 (LENA)
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