Modifications in the structure of the lichen Cladonia thallus in the aftermath of habitat contamination and implications for its heavy-metal accumulation capacity

Phenotypic traits of lichens can be greatly modified by environmental factors. Granulose thalli on soil and podetia, densely covered with granules, referring to common and widespread lichen Cladonia cervicornis subsp. verticillata were found near zinc smelter. The granules are stratified, filled with fungal medulla and heavily encrusted with calcium oxalate weddellite crystals, not observed on regularly developed thalli of the species. Phylogenetic analysis revealed that deformed granulose forms belong to this taxon, showing that the phenotypic plasticity of the lichens of Cladonia can lead to the emergence of features that do not coincide with the taxonomic definition of the species. The heavy-metal accumulation capacity of both granulose and regular form of primary and secondary lichen thallus, in relation to the element content in corresponding substrate, was determined. Granulose-modified thalli accumulate greater amounts of heavy metals than regular ones, meaning that the bioaccumulation property of a given species may be greatly affected by morphological modifications. The granulose forms are also characterised by considerably higher ratios of Cd, Pb and As concentrations in lichen samples in relation to the corresponding substrates than regular ones. This means that collection of variously formed thalli should be avoided in biomonitoring sampling procedures. The results indicate that a substantial part of the element load, in particular zinc, in the examined lichen thalli collected near the smelter originates from atmospheric fallout

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

High intraspecific genetic and morphological variation in the pioneer lichen Cladonia rei colonising slag dumps

This study investigates the genetic and morphological variability of the lichen Cladonia rei inhabiting strongly contaminated postsmelting slag dumps in southern Poland. Altogether, 27 C. rei samples were analysed, including 17 from a single population in one dump. The phylogenetic analysis includes samples of C. rei, outgroup species, and external sequences of Cladonia section representatives from GenBank. Comparative analysis of the internal transcribed spacer (ITS) rDNA sequences revealed the presence of 19 C. rei haplotypes overall, including several of the most frequent, of which 11 are represented by single individuals only. As many as 12 haplotypes were recorded within a single population. Three strongly supported monophyletic clades comprised of specimens from different geographical regions were recovered. Morphometric analysis showed great phenotypic variability within particular clades. Apart from a full range of previously known morphological forms of the species, an additional specific morphotype was recognised in the dumps; however, its representatives do not create a monophyletic group. High genetic variability within a single population suggests that C. rei has a great potential for colonising anthropogenic habitats. This attribute emphasises the role of this lichen as an essential pioneer in the early stages of natural regeneration of such sites

Intraspecific molecular variation of Allium ursinum (Amaryllidaceae) across the border of two subspecies distribution ranges

The study investigates the genetic differentiation among two subspecies of Allium ursinumL., namely A. ursinumsubsp. ursinumand subsp. ucrainicumas well as their putative hybrid that is represented by individuals withintermediate morphology. Inter-Simple Sequence Repeats (ISSR) were applied to determine the status of inter-mediate morphotypes in terms of their genetic pattern and to assess the level of genetic variability within andbetween various populations of A. ursinum. The study comprises 144 specimens from nine populations alongthe east-west transect in Poland, which includes localities of both subspecies and their putative hybrid. Amongthe examined populations, 48 bands were amplified, of which 45 were found to be polymorphic. The principalcoordinate analysis (PCoA), the neighbour-net analysis and Mantel test showed a strong correlation betweengenetic variability and geographic distance. Analysis of molecular variance (AMOVA) revealed that a greater pro-portion of total genetic variation resided within populations rather than among them. The Structure Bayesianclustering analysis revealed the presence of three distinct genetic groups within studied populations, where 'east-ern' genotypes correspond to A. ursinumsubsp. ucrainicum, and 'western' to subsp. ursinum; whereas the thirdgenetic group has the largest share in the individuals occurring at the border of the distribution ranges of bothsubspecies. The emergence of the third genetic group is probably an effect of hybridization events occurring with-in the secondary contact zone. Typical morphologically intermediate populations occur only in a relatively nar-row geographical zone, but the hybrid zone revealed by molecular markers is actually much wider than it is sug-gested by the morphological pattern of individuals. The current distribution pattern of both subspecies of A. ursinumand their hybrid zone is related to the two main directions of postglacial migration of Fagus syl-vaticato the area of Poland. The hybrid zone arose as an effect of the secondary contact of two divergent line-ages of A. ursinum

A biological study of composites based on the blends of nanohydroxyapatite, silk fibroin and chitosan

In this work, the biological properties of three-dimensional scaffolds based on a blend of nanohydroxyapatite (nHA), silk fibroin (SF), and chitosan (CTS), were prepared using a lyophilization technique with various weight ratios: 10:45:45, 15:15:70, 15:70:15, 20:40:40, 40:30:30, and 70:15:15 nHA:SF:CTS, respectively. The basic 3D scaffolds were obtained from 5% (w/w) chitosan and 5% silk fibroin solutions and then nHA was added. The morphology and physicochemical properties of scaffolds were studied and compared. A biological test was performed to study the growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). It was found that the addition of chitosan increases the resistance properties and extends the degradation time of materials. In vitro studies with human mesenchymal stem cells found a high degree of biotolerance for the materials produced, especially for the 20:40:40 and 15:70:15 (nHa:SF:CTS) ratios. The presence of silk fibroin and the elongated shape of the pores positively influenced the differentiation of cells into osteogenic cells. By taking advantage of the differentiation/proliferation cues offered by individual components, the composites based on the nanohydroxyapatite, silk fibroin, and chitosan scaffold may be suitable for bone tissue engineering, and possibly offer an alternative to the widespread use of collagen materials

Age and Skeletal Sites Affect BMP-2 Responsiveness of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) contain osteoprogenitors responsive to stimulation by osteogenic growth factors like bone morphogenetic proteins (BMPs). When used as grafts, BMSCs can be harvested from different skeletal sites such as axial, appendicular and orofacial bones, but the lower therapeutic efficacy of BMPs on BMSCs-responsiveness in humans compared to animal models may be partly due to effects of skeletal site and age of donor. We previously reported superior differentiation capacity and osteogenic properties of orofacial BMSCs relative to iliac crest BMSCs in same individuals. This study tested the hypothesis that recombinant human BMP-2 (rhBMP-2) stimulates human BMSCs differently based on age and skeletal site of harvest. Adult maxilla, mandible and iliac crest BMSCs from same individuals and pediatric iliac crest BMSCs were comparatively assessed for BMP-2 responsiveness under serum-containing and serum-free insulin-supplemented culture conditions. Adult orofacial BMSCs were more BMP-2-responsive than iliac crest BMSCs based on higher gene transcripts of alkaline phosphatase, osteopontin and osteogenic transcription factors MSX-2 and Osterix in serum-free insulin-containing medium. Pediatric iliac crest BMSCs were more responsive to rhBMP-2 than adult iliac crest BMSCs based on higher expression of alkaline phosphatase and osteopontin in serum-containing medium. Unlike orofacial BMSCs, MSX-2 and Osterix transcripts were similarly expressed by adult and pediatric iliac crest BMSCs in response to rhBMP-2. These data demonstrate that age and skeletal site-specific differences exist in BMSC osteogenic responsiveness to BMP-2 stimulation and suggest that MSX-2 and Osterix may be potential regulatory transcription factors in BMP-mediated osteogenesis of adult orofacial cells