Expression and function of ATP-dependent potassium channels in zebrafish islet β-cells

ATP-sensitive potassium channels (K(ATP) channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, K(ATP) channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism–secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet β-cells express functional K(ATP) channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish K(ATP) using diazoxide, a specific K(ATP) channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That β-cell K(ATP) channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes

Analysis of 2D airglow imager data with respect to dynamics using machine learning

We demonstrate how machine learning can be easily applied to support the analysis of large quantities of excited hydroxyl (OH*) airglow imager data. We use a TCN (temporal convolutional network) classification algorithm to automatically pre-sort images into the three categories “dynamic” (images where small-scale motions like turbulence are likely to be found), “calm” (clear-sky images with weak airglow variations) and “cloudy” (cloudy images where no airglow analyses can be performed). The proposed approach is demonstrated using image data of FAIM 3 (Fast Airglow IMager), acquired at Oberpfaffenhofen, Germany, between 11 June 2019 and 25 February 2020, achieving a mean average precision of 0.82 in image classification. The attached video sequence demonstrates the classification abilities of the learned TCN. Within the dynamic category, we find a subset of 13 episodes of image series showing turbulence. As FAIM 3 exhibits a high spatial (23 m per pixel) and temporal (2.8 s per image) resolution, turbulence parameters can be derived to estimate the energy diffusion rate. Similarly to the results the authors found for another FAIM station (Sedlak et al., 2021), the values of the energy dissipation rate range from 0.03 to 3.18 W kg−1

Analysis of 2D airglow imager data with respect to dynamics using machine learning

We demonstrate how machine learning can be easily applied to support the analysis of large quantities of excited hydroxyl (OH*) airglow imager data. We use a TCN (temporal convolutional network) classification algorithm to automatically pre-sort images into the three categories “dynamic” (images where small-scale motions like turbulence are likely to be found), “calm” (clear-sky images with weak airglow variations) and “cloudy” (cloudy images where no airglow analyses can be performed). The proposed approach is demonstrated using image data of FAIM 3 (Fast Airglow IMager), acquired at Oberpfaffenhofen, Germany, between 11 June 2019 and 25 February 2020, achieving a mean average precision of 0.82 in image classification. The attached video sequence demonstrates the classification abilities of the learned TCN. Within the dynamic category, we find a subset of 13 episodes of image series showing turbulence. As FAIM 3 exhibits a high spatial (23 m per pixel) and temporal (2.8 s per image) resolution, turbulence parameters can be derived to estimate the energy diffusion rate. Similarly to the results the authors found for another FAIM station (Sedlak et al., 2021), the values of the energy dissipation rate range from 0.03 to 3.18 W kg−1.</p

Unsaturated glycoceramides as molecular carriers for mucosal drug delivery of GLP-1

The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short- or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides

Insights on the trafficking and retro-translocation of glycosphingolipid-binding bacterial toxins

Some bacterial toxins and viruses have evolved the capacity to bind mammalian glycosphingolipids to gain access to the cell interior, where they can co-opt the endogenous mechanisms of cellular trafficking and protein translocation machinery to cause toxicity. Cholera toxin (CT) is one of the best-studied examples, and is the virulence factor responsible for massive secretory diarrhea seen in cholera. CT enters host cells by binding to monosialotetrahexosylganglioside (GM1 gangliosides) at the plasma membrane where it is transported retrograde through the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). In the ER, a portion of CT, the CT-A1 polypeptide, is unfolded and then “retro-translocated” to the cytosol by hijacking components of the ER associated degradation pathway (ERAD) for misfolded proteins. CT-A1 rapidly refolds in the cytosol, thus avoiding degradation by the proteasome and inducing toxicity. Here, we highlight recent advances in our understanding of how the bacterial AB5 toxins induce disease. We highlight the molecular mechanisms by which these toxins use glycosphingolipid to traffic within cells, with special attention to how the cell senses and sorts the lipid receptors. We also discuss several new studies that address the mechanisms of toxin unfolding in the ER and the mechanisms of CT A1-chain retro-translocation to the cytosol

Unsaturated glycoceramides as molecular carriers for mucosal drug delivery of GLP-1

The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short-or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides

Transcriptional changes in chick wing bud polarization induced by retinoic acid

BACKGROUND: Retinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies antero-posterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in antero-posterior patterning. To investigate this, we implanted retinoic acid-soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression. RESULTS: Retinoic acid upregulates expression of Hoxd11-13 that encode transcription factors implicated in inducing Shh transcription and that are involved in digit development. In our assay, retinoic acid induces Shh transcription and consequently a new pattern of digits at a much later stage than anticipated. Retinoic acid represses many anteriorly-expressed genes including Bmp4, Lhx9, Msx2 and Alx4. We provide evidence that retinoic acid influences transcription via induction of dHAND and inhibition of Gli3 to establish a new antero-posterior pre-pattern. We show that transient exposure to retinoic acid can suppress distal development and expedite cells to transcriptionally respond to Shh. CONCLUSIONS: Our findings reveal how retinoic acid and Shh signalling could co-operate in antero-posterior patterning of the limb. This article is protected by copyright. All rights reserved

Analysis of talpid3 and wild-type chicken embryos reveals roles for Hedgehog signalling in development of the limb bud vasculature

Chicken talpid mutant embryos have a wide range of Hedgehog-signalling related defects and it is now known that the talpid gene product encodes a novel protein essential for Hedgehog signalling which is required for both activator and repressor functions of Gli transcription factors (Davey, M.G., Paton, I.R., Yin, Y., Schmidt, M., Bangs, F.K., Morrice, D.R., Gordon-Smith, T., Buxton, P., Stamataki, D., Tanaka, M., Münsterberg, A.E., Briscoe, J., Tickle, C., Burt, D.W. (2006). The chicken talpid gene encodes a novel protein essential for Hedgehog signalling. Genes Dev 20 1365-77). Haemorrhaging, oedema and other severe vascular defects are a central aspect of the talpid phenotype (Ede, D.A. and Kelly, W.A (1964a). Developmental abnormalities in the head region of the talpid mutant fowl. J. Embryol. exp. Morp. 12:161-182) and, as Hedgehog (Hh) signalling has been implicated in every stage of development of the vascular system, the vascular defects seen in talpid are also likely to be attributable to abnormal Hedgehog signalling. Gene expression of members of the VEGF and Angiopoietin families of angiogenic growth factors has been linked to haemorrhaging and oedema and we find widespread expression of VEGF-D, rigf and Ang2a in the talpid limb. Furthermore, ectopic expression of these genes in talpid limbs points to regulation via Gli repression rather than activation. We monitored specification of vessel identity in talpid limb vasculature by examining expression of artery-specific genes, Np1 and EphrinB2, and the vein-specific genes, Np2a and Tie2. We show that there are supernumerary subclavian arteries in talpid limb buds and abnormal expression of an artery-specific gene in the venous submarginal sinus, despite the direction of blood flow being normal. Furthermore, we show that Shh can induce Np1 expression but has no effect on Np2a. Finally, we demonstrate that induction of VEGF and Ang2a expression by Shh in normal limb buds is accompanied by vascular remodelling. Thus Hedgehog signalling has a pivotal role in the cascade of angiogenic events in a growing embryonic organ which is similar to that proposed in tumours