Identification and Characterization of O-mannosylated Proteins

In mammals the O-mannosylation is a rare protein modification found only on proteins from muscles, brain and peripheral nerves. Although increased levels were detected in brain tissue only a few proteins have been identified to carry O mannosyl glycans so far. However, their O-mannosylation does not account for the high amount present in brain. In humans defects in the O-mannosylation pathway lead to severe malformations of muscles, eyes and brain revealing the importance of this modification. The pathogenic mechanism of these diseases, called dystroglycanopathies, was only analyzed for α-dystroglycan in more detail whose defective glycosylation can explain the muscle but not the brain phenotype. In this work new O-mannosylated proteins were identified in mammalian brain using an unbiased proteomics approach. Neurofascin isoform 186 from mouse brain was shown to carry O-mannosyl glycans as well as the lecticans brevican, neurocan and versican from murine and bovine brain. Thus, the O-mannosylation was shown to be similar among different mammalian species. Since the lecticans are highly expressed in brain, finally the high amount of O-mannosylation in brain can be explained. In addition, new insights into the pathogenic mechanism of dystroglycanopathies were gained. Because neurofascin and the lecticans play important roles in the stabilization of the extracellular matrix around neurons and in the establishment of specialized microdomains impairment of their functions by a defective O mannosylation might explain the brain-specific symptoms

Fundamental in vitro 3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection

Cytocompatibility Evaluation of a Novel Series of PEG-Functionalized Lactide-Caprolactone Copolymer Biomaterials for Cardiovascular Applications

Although the use of bioresorbable materials in stent production is thought to improve long-term safety compared to their durable counterparts, a recent FDA report on the 2-year follow-up of the first FDA-approved bioresorbable vascular stent showed an increased occurrence of major adverse cardiac events and thrombosis in comparison to the metallic control. In order to overcome the issues of first generation bioresorbable polymers, a series of polyethylene glycol-functionalized poly-L-lactide-co-ε-caprolactone copolymers with varying lactide-to-caprolactone content is developed using a novel one-step PEG-functionalization and copolymerization strategy. This approach represents a new facile way toward surface enhancement for cellular interaction, which is shown by screening these materials regarding their cyto- and hemocompatibility in terms of cytotoxicity, hemolysis, platelet adhesion, leucocyte activation and endothelial cell adhesion. By varying the lactide-to-caprolactone polymer composition, it is possible to gradually affect endothelial and platelet adhesion which allows fine-tuning of the biological response based on polymer chemistry. All polymers developed were non-cytotoxic, had acceptable leucocyte activation levels and presented non-hemolytic (<2% hemolysis rate) behavior except for PLCL-PEG 55:45 which presented hemolysis rate of 2.5% ± 0.5. Water contact angles were reduced in the polymers containing PEG functionalization (PLLA-PEG: 69.8° ± 2.3, PCL-PEG: 61.2° ± 7.5) versus those without (PLLA: 79.5° ± 3.2, PCL: 76.4° ± 10.2) while the materials PCL-PEG550, PLCL-PEG550 90:10 and PLCL-PEG550 70:30 demonstrated best endothelial cell adhesion. PLLA-PEG550 and PLCL-PEG550 70:30 presented as best candidates for cardiovascular implant use from a cytocompatibility perspective across the spectrum of testing completed. Altogether, these polymers are excellent innovative materials suited for an application in stent manufacture due to the ease in translation of this one-step synthesis strategy to device production and their excellent in vitro cyto- and hemocompatibility

Bringing Neuroscientific Data to Sustainability: Embedded Data Stewardship in CRC 1280

Information Management (INF) projects are essential to the establishment of infrastructures for the management of research data and metadata in Collaborative Research Centers (CRCs) funded by the German Research Foundation (DFG). However, the CRC's disciplines, the INF project's design, and the institutional setting all have a significant impact on the organization and daily responsibilities of INF data stewards. We demonstrate the impact of embedded data stewardship on research data management (RDM) in the case of CRC 1280 "Extinction Learning". Central aim of the RDM in CRC 1280 was the implementation of data sharing across all subprojects at an early stage of the research life cycle. Despite obstacles such as legal issues regarding sensitive data and a highly competitive culture in life science, the CRC was successful in realising this goal. Key to this success was building trust with researchers through participatory strategy development, the implementation of supportive training formats and personal, on-site consultations by an embedded data steward with appropriate scientific background. In addition, the CRC’s endeavours were led to sustainability by bridging the gap between researchers and central infrastructure. INF paved the way for the CRC to participate in the development of the university-central repository infrastructure, enabling the emergence of a sustainable RDM infrastructure tailored to the needs of the researchers