The Granuphilin homolog bitesize regulates sleep and longevity in Drosophila melanogaster

In a RNAseq based screen of long-lived dilp 2-3,5 mutants, the btsz locus was identified as a potential downstream effector. The function of Granuphilin, the mammalian homolog of btsz, has been well described to mediate the exocytosis of insulin-containing granules in pancreatic beta cells via interaction with Rab27a (Yi et al. 2002, Torii et al. 2004, Gomi et al. 2005). In contrast, the function of Btsz, specifically the P1 isoform containing the highly conserved SHD domain, is barely described in Drosophila. A previous study conducted in our lab revealed, that Btsz P1 is exclusively expressed in the mushroom body (Weigelt 2018), a brain region that has been implicated in the regulation of sleep and olfactory learning (Joiner et al. 2006). Strikingly, deletion of btsz P1 resulted in a significant lifespan extension in male and female flies and increased sleep length in young male flies (Weigelt 2018). In this study we showed that deletion of btsz P1 significantly influenced sleep patterns in both, male and female flies, in line with the observed lifespan extension in both sexes. Notably, btsz P1∆ mutants also exhibited an improved sleep quality during ageing, which was accompanied by ameliorated deterioration in locomotion during ageing, indicating that btsz P1∆ mutants do not only live longer, but also show a delayed onset of a characteristic physiological decline. Since btsz P1∆ mutants phenocopy dilp 2-3,5 mutants in extended lifespan, reduced fecundity and weight, and prolonged nocturnal sleep, we wondered if Btsz P1 acts on the insulin-signalling pathway. Therefore, we performed genetic epistasis experiments and combined both mutations. The combination of both mutations, however, did not result in additive effects regarding lifespan and sleep, but rather resulted in detrimental effects. In addition, Btsz P1 did not localize in insulin-secreting neurons, suggesting that Btsz P1 influences insulin-signalling probably indirectly. To further investigate whether Btsz P1 regulates other sleep-regulating pathways, we modulated several sleep-regulating signalling-pathways pharmacologically and genetically. Sleep analysis revealed that btsz P1∆ mutants are resistant to interventions on the dopamine-signalling pathway, implying that Btsz P1 potentially acts in dopaminergic neurotransmission. Brain immunostainings revealed that Btsz P1 is not expressed in dopaminergic neurons, but in neurons expressing DopR, further supporting our sleep data. Also, analysis of the single cell expression atlas of the ageing Drosophila brain strengthened our hypothesis that Btsz P1 probably executes its function in neurons expressing DopR, since expression of btsz P1 was detected in the same neuronal clusters as DopR. Interestingly, Ca-alpha 1T, a channel that has been implicated in the regulation of sleep, colocalised with Btsz P1 in neurons expressing DopR. In addition, Ca-alpha 1T null mutants were resistant to methamphetamine treatment, reminiscent to btsz P1∆ mutants, rising the hypothesis that Btsz P1 might influence the exocytosis of Ca-alpha 1T to the transmembrane to restore-ion currencies in order to regulate the neuronal excitability

Design of a parallel robot with additively manufactured flexure hinges for a cryogenic work environment

Automation is ubiquitous in today's industrial landscape and is finding its way into more and more highly specialised applications - also in the field of cryopreservation. The extreme work conditions in cryobanks place exceptionally high demands on the mechanical and electronic components used. The preservation and storage of biological samples take place at temperatures between -130 °C and -196 °C using liquid nitrogen as a cooling medium. The bearings and joints used in industrial parallel kinematic robots (for example, ball bearings or Cardan joints) jam at these ambient parameters and are unsuitable for an application within a cryobank. We, therefore, develop methods and technologies to enable fully automated handling of biological samples under cryogenic working conditions. The basis for this is a parallel kinematic robot structure that allows the drives to be placed outside the cold environment. In contrast, the rest of the robot structure can be actuated in a cryogenic container. In this context, the passive joints for this parallel robot are designed as additively manufactured monolithic flexure hinges. This paper presents the design, simulation, and construction of the parallel robot and focuses on the flexure hinges fabricated using the selective laser melting process (SLM). We describe the design of the flexure hinges, their intended use in the robot, and the experimental setup used for their validation. We also compare the operating parameters recorded in experiments (such as bending angle, bending moment) with the data obtained in finite element method simulations (FEM). In addition, we describe the geometric constraints and deviations of the manufactured joints due to the manufacturing process

Combined Structural and Dimensional Synthesis of a Parallel Robot for Cryogenic Handling Tasks

The combined structural and dimensional synthesis is a tool for finding the robot structure that is suited best for a given task by means of global optimization. The handling task in cryogenic environments gives strong constraints on the robot synthesis, which are translated by an engineering design step into the combined synthesis algorithm. This allows to reduce the effort of the combined synthesis, which provides concepts for alternative robot designs and indications on how to modify the existing design prototype, a linear Delta robot with flexure hinges. Promising design candidates are the 3PRRU and 3PRUR, which outperform the linear Delta (3PUU) regarding necessary actuator force

Numerical simulation and statistical analysis of a cascaded flexure hinge for use in a cryogenic working environment

Due to their many advantages, flexible structures are increasingly being used as guide and transmission elements in handling systems. Prismatic solid-state joints with a concentrated cross-sectional reduction are predominantly used as flexure pivots for both microscopic and macroscopic designs. A transfer of these geometries to applications in cryogenic working environments is not easily possible at temperatures below -130 °C due to the changed material properties. In this paper, the further development of swivel joints as cascaded solid state joints for such a cryogenic environment is illustrated by the targeted adaptation of certain joint parameters and dimensions. By means of a comprehensive FEM simulation, it can be shown how the influence of specific parameters affects movement accuracy, process forces and shape stability and to what extent these geometric parameters influence each other in their effect

Physical Self-Concept and Physical Activity in Children with Congenital Heart Defects—Can We Point Out Differences to Healthy Children to Promote Physical Activity?

Objective: Children with congenital heart defects (CHD) are at high risk for cardiovascular disease in addition to their congenital disease, so it is important to motivate this group of patients to live a physically active lifestyle. A potential influencing determinant of younger children’s physical performance is the physical self-concept. The objective of the present study was first to evaluate the correlation between the physical self-concept (PSC) and the participation in physical activities (PA) of a representative group of children with congenital heart disease (CHD), and second to point out differences in comparison to their healthy peer group. Methods: Using the database of PA of the S-BAHn-Study we focused on physical self-concept assessed by the German version of the Physical Self-Description Questionnaire. We compare the obtained data of children with CHD to a representative age-matched sample of 3.385 participants of the Motorik Modul Study. Results: N = 1.198 complete datasets could be included in the analyses. The mean age of patients was 11.6 ± 3.1 years. For the total cohort of patients with CHD and the reference group, PA correlated significantly with a positive PSC (p 0.24). Conclusions: According to this representative survey, there is a clear relation between PA and PSC in the cohort of healthy children and the group of children with CHD throughout the severity of their heart defects. Although PSC did not differ in patients with simple CHD and their healthy peer group, PA was significantly reduced. This gap invites us to reflect on how we could break new ground to promote a physically active lifestyle in children with CHD regardless of the severity of their cardiac defects

Induktive Energieübertragung in eine kryogene Umgebung : Design und Charakterisierung einer drahtlosen Energieübertragungsstrecke für den Betrieb einer Greiferaktorik

In der Forschung und Industrie ist die Automatisierung allgegenwärtig und findet ihren Weg in immer mehr hochspezialisierte Anwendungen - einschließlich der Kryokonservierung. Dennoch ist die manuelle Handhabung von biologischen oder toxischen Proben in wissenschaftlichen und kommerziellen Lagereinrichtungen immer noch vorherrschend. Dies bedingt für das Personal ein erhebliches Verletzungsrisiko durch Kälteverbrennungen. Darüber hinaus wird die Unversehrtheit der Proben durch Temperaturschwankungen oder Verunreinigungen gefährdet. In diesem Beitrag wird ein Ansatz für die Automatisierung von Handhabungsprozessen bei tiefen Tem-peraturen zwischen -130 °C und -190 °C in Kryobanken vorgestellt. Das Automatisierungssystem basiert auf einem Parallelroboter, da seine Struktur die Positionierung der Antriebe außerhalb des gekühlten Arbeitsraums erlaubt. Die Gelenke und Manipulatoren des Roboters befinden sich innerhalb des kroygenen Lagerbehälters, der mit flüssigem Stickstoff gekühlt wird. Die Energieversorgung der kryotauglichen Greiferaktorik im Inneren des Lagerbehälters erfolgt induktiv. Zu diesem Zweck wurden Varianten von Spulendesigns und deren Anordnung mit der FEM-Software ANSYS unter Einbeziehung von applikationsspezifischen Randbedingungen modelliert. Die Dimensionierung der Schwingkreise wurde mit Berechnungen in Mathcad ergänzend durchgeführt. Die entwickelte Greiferaktorik lässt sich in flüssigem Stickstoff mit einem Wirkungsgrad von etwa 86% bei einem Spulen-abstand von 9,5 cm und noch mit etwa 10% Wirkungsgrad bei einem Spulenabstand von 33,5 cm betreiben

Roboterkomponenten für den kryogenen Arbeitsraum : Entwicklung von Festkörpergelenken und monolithischen Greifersystemen für eine Parallelroboterstruktur

In der heutigen Industrie ist die Automatisierung ein allgegenwärtiger Faktor, selbst in Nischenanwendungen wie der Kryokonservierung. Die manuelle Handhabung von biologischen oder toxischen Proben ist in Forschungseinrichtungen immer noch die Norm. Die Konservierung und Lagerung solcher Proben erfolgt in sogenannten Kryobanken bei Temperaturen zwischen -130 °C und -196 °C. In heute üblichen Kryobanken werden die Proben oft mit sperriger Schutzkleidung von Hand ein- und ausgelagert oder bewegt. Dies ist notwendig, da ein erhebliches Verletzungsrisiko für den Arbeiter durch Kälteverbrennungen sowie eine Gefährdung der Probenintegrität durch Beschädigung und Erwärmung oder auch Temperaturwechsel besteht. Zur Überwindung dieser Probleme ist eine Vollautomatisierung bei Temperaturen unter -130 °C wünschenswert. In diesem Beitrag wird das von der DFG geförderte Projekt "Methoden zur Automatisierung von Handhabungsprozessen unter kryogenen Umgebungs-bedingungen" vorgestellt und erläutert. Dabei wird besonders auf die Teilaspekte der Greifertechnologie sowie der Optimierung der verwendeten Festkörpergelenke eingegangen

Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity

G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPRs) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPRs have shown that arginine-rich DPRs are most toxic, while polyGlycine–Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length or tagging affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in patients

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions