Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis.

BACKGROUND: Upregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities. METHODS: NRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines. RESULTS: HTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines. CONCLUSIONS: Contrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.

Establishment of the basidiomycete Fomes fomentarius for the production of composite materials.

BACKGROUND: Filamentous fungi of the phylum Basidiomycota are considered as an attractive source for the biotechnological production of composite materials. The ability of many basidiomycetes to accept residual lignocellulosic plant biomass from agriculture and forestry such as straw, shives and sawdust as substrates and to bind and glue together these otherwise loose but reinforcing substrate particles into their mycelial network, makes them ideal candidates to produce biological composites to replace petroleum-based synthetic plastics and foams in the near future. RESULTS: Here, we describe for the first time the application potential of the tinder fungus Fomes fomentarius for lab-scale production of mycelium composites. We used fine, medium and coarse particle fractions of hemp shives and rapeseed straw to produce a set of diverse composite materials and show that the mechanical materials properties are dependent on the nature and particle size of the substrates. Compression tests and scanning electron microscopy were used to characterize composite material properties and to model their compression behaviour by numerical simulations. Their properties were compared amongst each other and with the benchmark expanded polystyrene (EPS), a petroleum-based foam used for thermal isolation in the construction industry. Our analyses uncovered that EPS shows an elastic modulus of 2.37 ± 0.17 MPa which is 4-times higher compared to the F. fomentarius composite materials whereas the compressive strength of 0.09 ± 0.003 MPa is in the range of the fungal composite material. However, when comparing the ability to take up compressive forces at higher strain values, the fungal composites performed better than EPS. Hemp-shive based composites were able to resist a compressive force of 0.2 MPa at 50% compression, rapeseed composites 0.3 MPa but EPS only 0.15 MPa. CONCLUSION: The data obtained in this study suggest that F. fomentarius constitutes a promising cell factory for the future production of fungal composite materials with similar mechanical behaviour as synthetic foams such as EPS. Future work will focus on designing materials characteristics through optimizing substrate properties, cultivation conditions and by modulating growth and cell wall composition of F. fomentarius, i.e. factors that contribute on the meso- and microscale level to the composite behaviour.

Development of Polythiourethane/ZnO-Based Anti-Fouling Materials and Evaluation of the Adhesion of Staphylococcus aureus and Candida glabrata Using Single-Cell Force Spectroscopy.

The attachment of bacteria and other microbes to natural and artificial surfaces leads to the development of biofilms, which can further cause nosocomial infections. Thus, an important field of research is the development of new materials capable of preventing the initial adhesion of pathogenic microorganisms. In this work, novel polymer/particle composite materials, based on a polythiourethane (PTU) matrix and either spherical (s-ZnO) or tetrapodal (t-ZnO) shaped ZnO fillers, were developed and characterized with respect to their mechanical, chemical and surface properties. To then evaluate their potential as anti-fouling surfaces, the adhesion of two different pathogenic microorganism species, Staphylococcus aureus and Candida glabrata, was studied using atomic force microscopy (AFM). Our results show that the adhesion of both S. aureus and C. glabrata to PTU and PTU/ZnO is decreased compared to a model surface polydimethylsiloxane (PDMS). It was furthermore found that the amount of both s-ZnO and t-ZnO filler had a direct influence on the adhesion of S. aureus, as increasing amounts of ZnO particles resulted in reduced adhesion of the cells. For both microorganisms, material composites with 5 wt.% of t-ZnO particles showed the greatest potential for anti-fouling with significantly decreased adhesion of cells. Altogether, both pathogens exhibit a reduced capacity to adhere to the newly developed nanomaterials used in this study, thus showing their potential for bio-medical applications.