hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased β-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.     

Relationship between crystalline structure and mechanical behavior in isotropic and oriented polyamide 6

Polyamide 6 (PA6) isotropic films and oriented cables were prepared by compression molding or by consecutive extrusion and cold‐drawing. These samples were isothermally annealed in the 120–200°C range and were then subjected to tensile tests at room temperature. Synchrotron wide‐angle X‐ray scattering (WAXS) and small‐angle X‐ray scattering (SAXS) patterns were obtained before and after mechanical failure. These data were related with the mechanical properties of the respective PA6 samples. The annealing of isotropic PA6 resulted in an increase in the Young's modulus (E) and yield stress (σ_y) values, which was attributed to the observed proportional reduction of the d‐spacings of the intersheet distances in both the α‐PA6 and γ‐PA6 polymorphs. Analysis of the WAXS and SAXS patterns of isotropic PA6 after break allowed the supposition of structural changes in the amorphous phase, with these being better pronounced with increasing annealing temperature; this made the samples less ductile. In oriented PA6 samples, annealing resulted in a drastic increase in the E and σ_y values accompanied by a phase transition from γ‐PA6 to α‐PA6 and a well‐pronounced reduction in the intersheet distances of both polymorphs. The stretching of the oriented samples led to an additional γ‐to‐α transition, whose extent was also related to structural changes in the amorphous phase. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2242–2252, 2007     

Radical‐scavenging properties of extracts from the white button mushroom,Agaricus bisporus

BACKGROUND: Agaricus bisporus is the premier cultivated edible mushroom but is usually considered to be of lesser value nutritionally and medicinally compared with other cultivated mushrooms. The objective of this study was to investigate the radical‐scavenging properties of methanolic extracts and the free radical‐processing enzyme activities of water extracts from A. bisporus fruit bodies. Analyses were performed on total fruit bodies from three strains and on separated stipe, cap and gills from one strain, all cultivated experimentally under the same conditions. RESULTS: EC₅₀ values of scavenging ability on 1,1‐diphenyl‐2‐picrylhydrazyl radicals (DPPH^?), scavenging ability on 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cations (ABTS^?+) and reducing power of methanolic extracts from total fruit bodies ranged between 1.7 and 5.0 and were 1.1–1.9 times higher in a white hybrid than in two wild strains, cream and brown. The gills exhibited higher antioxidant activities relative to the stipe and cap. In water extracts, glutathione peroxidase activities were up to eight times higher than glutathione reductase activities and no difference was observed between strains. Catalase activity was highest in the brown wild strain. The only significant differences in free radical‐processing enzymes between the three parts of the mushroom were higher catalase activity in the gills and lower glutathione reductase activity in the stipe. CONCLUSION: The radical‐scavenging properties of the button mushroom are comparable to those of other edible mushrooms and dependent on the strain and on the section of the fruit body. Copyright © 2008 Society of Chemical Industry     

Preparation and properties of polyamide‐6‐based thermoplastic laminate composites by a novel in‐mold polymerization technique

In this work, a method for preparation of polyamide‐6 (PA6) based laminates reinforced by glass fiber‐ (GFL) or polyamide‐66 (PA66) textile structures (PL) via reactive injection molding is disclosed. It is based on in‐mold anionic polymerization of ε‐caprolactam carried out at 165°C in the presence of the respective reinforcements performed in newly developed prototype equipment whose design concept and operation are described. Both composite types were produced for reaction times of 20 min, with conversion degrees of 97–99%. Initial mechanical tests in tension of GFL samples displayed almost twofold increase of the Young's modulus and stress at break values when compared with the neat anionic PA6. The improvement was proportional to the volume fraction V_f of glass fiber fabric that was varied in the 0.16–0.25 range. A 300% growth of the impact strength was registered in PL composites with V_f of PA66 textile of 0.1. Removing the surface finish of the latter was found to be a factor for improving the adhesion at the matrix–fiber interface. The mechanical behavior of GFL and PL composites was discussed in conjunction with the morphology of the samples studied by optical and electron microscopy and the matrix crystalline structure as revealed by synchrotron X‐ray diffraction. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40083.     

Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of the mushrooms Agaricus bisporus and Pleurotus spp.

Hydrogen peroxide is suspected of being highly implicated in mushroom nutrition and in substrate bleaching during cultivation. The parameters for measuring H₂O₂ in compost samples were examined and the methodology was applied to samples from both compost colonized by cultivars and wild isolates of Agaricus bisporus, and wheat straw or coffee pulp colonized by Pleurotus spp. Laccase and peroxidase activities were also measured. H₂O₂ concentration measured after heating at 80 °C for inactivating laccases and peroxidases was probably both H₂O₂ pre‐existing in the compost and H₂O₂ generated from quinones and active oxygen species. This potential H₂O₂ concentration increased during the vegetative growth for all the strains, in agreement with a direct relationship between H₂O₂ concentration and active biomass of A. bisporus or Pleurotus spp. in their cultivation substrates. Correlations were observed between H₂O₂ concentration and manganese peroxidase activity in cultivation substrates at the stage of primordia formation. At this stage of development, H₂O₂ generation via biotic or abiotic mechanisms should be an important physiological trait of mushrooms. Copyright © 2007 Society of Chemical Industry