2016, Davies, Heather S., Singh, Prabha, Deckert-Gaudig, Tanja, Deckert, Volker, Rousseau, Karine, Ridley, Caroline E., Dowd, Sarah E., Doig, Andrew J., Pudney, Paul D. A., Thornton, David J., Blanch, Ewan W.

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.

2014, Appelhans, Dietmar, Klajnert-Maculewicz, Barbara, Janaszewska, Anna, Lazniewska, Joanna, Voit, Brigitte

In this review we highlight the potential for biomedical applications of dendritic glycopolymers based on polyamine scaffolds. The complex interplay of the molecular characteristics of the dendritic architectures and their specific interactions with various (bio)molecules are elucidated with various examples. A special role of the individual sugar units attached to the dendritic scaffolds and their density is identified, which govern ionic and H-bond interactions, and biological targeting, but to a large extent are also responsible for the significantly reduced toxicity of the dendritic glycopolymers compared to their polyamine scaffolds. Thus, the application of dendritic glycopolymers in drug delivery systems for gene transfection but also as therapeutics in neurodegenerative diseases has great promise.

2016, Thamm, Kristina, Graupner, Sylvi, Werner, Carsten, Huttner, Wieland B., Corbeil, Denis, Nabi, Ivan R

The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

2014, Kasten, Annika, Naser, Tamara, Brüllhoff, Kristina, Fiedler, Jörg, Müller, Petra, Möller, Martin, Rychly, Joachim, Groll, Jürgen, Brenner, Rolf E., Engler, Adam J.

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.

2015, Beer, Meike V., Hahn, Kathrin, Diederichs, Sylvia, Fabry, Marlies, Singh, Smriti, Spencer, Steve J., Salber, Jochen, Möller, Martin, Shard, Alexander G., Groll, Jürgen

Hydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.

2017, Bachhuka, Akash, Delalat, Bahman, Ghaemi, Soraya Rasi, Gronthos, Stan, Voelcker, Nicolas H., Vasilev, Krasimir

Advanced medical devices, treatments and therapies demand an understanding of the role of interfacial properties on the cellular response. This is particularly important in the emerging fields of cell therapies and tissue regeneration. In this study, we evaluate the role of surface nanotopography on the fate of human dental pulp derived stem cells (hDPSC). These stem cells have attracted interest because of their capacity to differentiate to a range of useful lineages but are relatively easy to isolate. We generated and utilized density gradients of gold nanoparticles which allowed us to examine, on a single substrate, the influence of nanofeature density and size on stem cell behavior. We found that hDPSC adhered in greater numbers and proliferated faster on the sections of the gradients with higher density of nanotopography features. Furthermore, greater surface nanotopography density directed the differentiation of hDPSC to osteogenic lineages. This study demonstrates that carefully tuned surface nanotopography can be used to manipulate and guide the proliferation and differentiation of these cells. The outcomes of this study can be important in the rational design of culture substrates and vehicles for cell therapies, tissue engineering constructs and the next generation of biomedical devices where control over the growth of different tissues is required.

2017, Bekeschus, Sander, Bräutigam, Lars, Wende, Kristian, Hanschmann, Eva-Maria

[No abstract available]

2018, Gutowska-Owsiak, Danuta, de La Serna, Jorge Bernardino, Fritzsche, Marco, Naeem, Aishath, Podobas, Ewa I., Leeming, Michael, Colin-York, Huw, O’Shaughnessy, Ryan, Eggeling, Christian, Ogg, Graham S.

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.

2018, Steuer, Anna, Wolff, Christina M., von Woedtke, Thomas, Weltmann, Klaus-Dieter, Kolb, Juergen F.

Pulsed electric fields (PEFs) and cold atmospheric pressure plasma (CAP) are currently both investigated for medical applications. The exposure of cells to PEFs can induce the formation of pores in cell membranes and consequently facilitate the uptake of molecules. In contrast, CAP mainly acts through reactive species that are generated in the liquid environment. The objective of this study was to determine, if PEFs combined with plasma-treated cell culture medium can mutually reinforce effects on viability of mammalian cells. Experiments were conducted with rat liver epithelial WB-F344 cells and their tumorigenic counterpart WB-ras for a direct comparison of non-tumorigenic and tumorigenic cells from the same origin. Viability after treatments strongly depended on cell type and applied field strength. Notably, tumorigenic WB-ras cells responded more sensitive to the respective treatments than non-tumorigenic WB-F344 cells. More cells were killed when plasma-treated medium was applied first in combination with treatments with 100-μs PEFs. For the reversed treatment order, i.e. application of PEFs first, the combination with 100-ns PEFs resulted in a stimulating effect for non-tumorigenic but not for tumorigenic cells. The results suggest that other mechanisms, besides simple pore formation, contributed to the mutually reinforcing effects of the two methods.

2015, Schmidt, Anke, Dietrich, Stephan, Steuer, Anna, Weltmann, Klaus-Dieter, von Woedtke, Thomas, Masur, Kai, Wende, Kristian

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.