A role for Egfl7 during endothelial organization in the embryoid body model system

Epidermal growth factor-like domain 7, Egfl7, is a largely endothelial restricted gene which is thought to have a role during the differentiation of embryonic stem cells (ESCs) along the endothelial lineage. While it has been shown that Egfl7 knock-down in zebrafish impairs endothelial cord formation, the role of the gene in mammals has been unresolved. Interpretation of mouse knockout studies has been complicated by the fact that deletion of miR-126, an intronic microRNA located within Egfl7, results in vascular defects. Here we use an siRNA knock-down approach to target specific regions of Egfl7 without affecting miR-126 expression. Egfl7 was knocked down in mouse ESCs and the effect on vascular development was assessed using the in vitro embryoid body (EB) model after either 7 or 14 days of differentiation. Knock-down of Egfl7 resulted in the formation of abnormal sheet-like CD31+ structures that were abundant within EBs after 7 days of differentiation. Only up to 60% of these sheets co-expressed basement membrane and endothelial cell junction markers. Similar CD31+ sheets were also seen as outgrowths from 7 day EBs into collagen gels. A partial remodelling occurred by 14 days of differentiation when fewer CD31+ sheets were seen both within EBs, and as outgrowths from EBs. Formation of these sheets was due, at least in part, to increased proliferation specifically of CD31+ cells. Cell death within EBs was unaffected by Egfl7 knock-down. In conclusion, our work shows that knock-down of Egfl7 causes defects in early vascular cord formation, and results in the development of CD31+ sheet-like structures. This suggests that Egfl7 is vital for the formation of endothelial cell cords, and that the gene has an important role during both vasculogenesis and angiogenesis in mammalian cells

LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion

Glucose homeostasis depends critically on insulin that is secreted by pancreatic β-cells. Serum glucose, which is directly sensed by β-cells, stimulates depolarization- and Ca(2+)-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels. Hypotonicity- or glucose-induced β-cell swelling elicits canonical LRRC8A-dependent VRAC currents that depolarize β-cells to an extent that causes electrical excitation. Glucose-induced excitation and Ca(2+) responses are delayed in onset, but not abolished, in β-cells lacking the essential VRAC subunit LRRC8A. Whereas Lrrc8a disruption does not affect tolbutamide- or high-K(+)-induced insulin secretion from pancreatic islets, it reduces first-phase glucose-induced insulin secretion. Mice lacking VRAC in β-cells have normal resting serum glucose levels but impaired glucose tolerance. We propose that opening of LRRC8/VRAC channels increases glucose sensitivity and insulin secretion of β-cells synergistically with K(ATP) closure. Neurotransmitter-permeable LRRC8D-containing VRACs might have additional roles in autocrine/paracrine signaling within islets

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Die Fackel. Herausgeber: Karl Kraus. Nr. 1 (1899) - Nr. 922 (1936). Volltextausgabe mit Bibliografie undRegister von Wolfgang Hink. CD- ROM-Edition herausgegeben von Friedrich Pfäffli

A New Mathematical Programming Formulation for the Single-Picker Routing Problem in a Single-Block Layout

The Single-Picker Routing Problem deals with the determination of sequences according to which items have to be picked in a distribution warehouse and the identification of the corresponding paths which have to be travelled by human operators (order pickers). The Single-Picker Routing Problem represents a special case of the classic Traveling Salesman Problem (TSP) and, therefore, can also be modeled as a TSP. However, the picking area of a warehouse typically possesses a block layout, i.e. the items are located in parallel picking aisles, and the order pickers can only change over to another picking aisle at certain positions by means of so-called cross aisles. In this paper, for the first time a mathematical programming formulation is proposed which takes into account this specific property. Based on extensive numerical experiments, it is shown that the proposed formulation is superior to standard TSP formulations

Amigo2 - a novel neuroprotective molecule produced by microglia cells

Mikroglia ist eine im zentralen Nervensystem (ZNS) ansässige, immunologisch aktive Zellgruppe, deren Aufgabe es ist, Nervenzellen zu schützen und funktionsfähig zu halten. Entzündungen im ZNS werden meist als ein zerstörerischer Prozess angesehen, der zu Schäden im Gewebe von Hirn und Rückenmark führt. Dabei wird häufig nicht berücksichtigt, dass eine Entzündungsreaktion von Nöten ist, um eine angemessene Heilung und Regeneration des Gewebes zu entfachen. Während einer durch TH1-Zellen ausgelösten Entzündung, kommt es zur verstärkten Ausschüttung des proinflammatorischen Zytokins Interferon-γ (IFN-γ), welches die Mikroglia Zellen aktiviert. Frühere Literaturhinweise belegten, dass TH1-Zell vermittelte Entzündungsreaktionen positive Auswirkungen auf die Nervenregeneration nach Rückenmarksverletzungen haben. Können diese Auswirkungen auf Faktoren zurückgeführt werden, die von IFN-γ aktivierten Mikroglia Zellen produziert werden? Um diese Frage zu beantworten, haben wir Mikroglia-Kulturen mit IFN-γ behandelt und nach einem veränderten Expressionsmuster von neuroprotektiven Molekülen gesucht. Dabei zeigte sich, dass ein Adhäsionsmolekül namens Amigo2 hochreguliert wird. Wir konnten zeigen, dass eine substratgebundene Form der extrazellulären Domäne des rekombinanten Proteins in hippokampalen Neuronen-Kulturen das Auswachsen von Neuriten induziert. Die Zugabe desselben rekombinanten Proteins in den Überstand von Mikroglia-Kulturen führt zu einer Veränderung der Adhäsionseigenschaften dieser Zellen. Eine Behandlung von Mikroglia Zellen mit IFN-γ führte auch zu einer geringeren Expression des Proteins Osteopontin. Zugabe rekombinanten Osteopontins in das Medium von Neuronen-Kulturen führt zu einer teilweisen Unterdrückung des Auswuchses der Neuriten. Diese beiden Befunde belegen eindeutig, dass IFN-γ behandelte Mikroglia Zellen dazu beitragen können, das Auswachsen von Nervenfasern nach Rückenmarksverletzungen zu fördern.Microglia are the resident immunocompetent cells of the central nervous system (CNS) and their role is to support and maintain the proper function of neuronal cells. Inflammation is often considered as a destructive process and a mediator of neuropathology, but it is also needed to induce an appropriate healing response. During a TH1-cell driven inflammation, the proinflammatory cytokine Interferon-γ (IFN-γ) is released, which leads to an activation of microglia cells, which precedes the action of the other cell types. Former reports have shown that a TH1-cell based inflammatory reaction has a beneficial effect on the recovery of spinal cord injuries (SCI). This raised the question, whether this positive outcome may be based on factors produced by IFN-γ activated microglia cells. To address this point, we treated microglia cell cultures with IFN-γ and looked for a differential expression of potential neuroprotective molecules. We found an adhesion molecule named Amigo2 to be upregulated. Coating of the recombinant extracellular domain of Amigo2 on culture dishes induced neurite outgrowth in hippocampal neurons, and in solution, the protein interfered with the adhesion properties of microglia cells. We also found Osteopontin (SPP1) to be prominently downregulated in IFN-γ primed microglia. The addition of Osteopontin into the medium of hippocampal cultures partly inhibited neurite outgrowth. We therefore suggest that the differential expression of those two molecules in IFN-γ treated cells can contribute in promoting the regeneration of spinal cord nerves after injury