Upper gut heat shock proteins HSP70 and GRP78 promote insulin resistance, hyperglycemia, and non-alcoholic steatohepatitis.

A high-fat diet increases the risk of insulin resistance, type-2 diabetes, and non-alcoholic steato-hepatitis. Here we identified two heat-shock proteins, Heat-Shock-Protein70 and Glucose-Regulated Protein78, which are increased in the jejunum of rats on a high-fat diet. We demonstrated a causal link between these proteins and hepatic and whole-body insulin-resistance, as well as the metabolic response to bariatric/metabolic surgery. Long-term continuous infusion of Heat-Shock-Protein70 and Glucose-Regulated Protein78 caused insulin-resistance, hyperglycemia, and non-alcoholic steato-hepatitis in rats on a chow diet, while in rats on a high-fat diet continuous infusion of monoclonal antibodies reversed these phenotypes, mimicking metabolic surgery. Infusion of these proteins or their antibodies was also associated with shifts in fecal microbiota composition. Serum levels of Heat-Shock-Protein70 and Glucose-Regulated Protein78were elevated in patients with non-alcoholic steato-hepatitis, but decreased following metabolic surgery. Understanding the intestinal regulation of metabolism may provide options to reverse metabolic diseases.

Production of natural colorants by liquid fermentation with Chlorociboria aeruginascens and Laetiporus sulphureus and prospective applications.

The replacement of potentially hazardous synthetic dyes with natural dyes and pigments are of great interest for a sustainable economy. In order to obtain cost-efficient, environmentally friendly and competitive products, improvements in the cultivation and extraction of pigment-producing organisms and in dyeing processes are necessary. In our study, we were able to scale up the production of xylindein by Chlorociboria aeruginascens from 3 to 70 L bioreactor cultivations. We have identified important bioprocess parameters like low shear stress (150 rpm, tip speed <0.5 m/s) for optimal pigment yield (4.8 mg/L/d). Additionally, we have demonstrated the potential of laetiporic acid production by Laetiporus sulphureus in various cultivation systems and media, achieving dried biomass concentrations of almost 10 g/L with a 7 L bioreactor cultivation after 17 days. Extractions performed at 70°C and 15 min incubation time showed optimal results. To the best of our knowledge, we have described for the first time the use of this pigment in silk dyeing, which results in a brilliant hue that cannot easily be produced by other natural pigments.

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy.

Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy - a fast, real-time, and label-free bio-sensing method - represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme-linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target-monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme-linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target-layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance-based system for continuous process monitoring.

Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis.

The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling.

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001.

Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/ß-catenin pathway in a Drosophila model for intellectual disability.

We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of ß-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/ß-catenin pathway possibly by enhancing the degradation of dsh.