Fabrication and Characterization of Protein Arrays for Stem Cell Patterning

Microarrays of fibronectin and other extracellular matrix (ECM) proteins were fabricated on plasmadeposited poly(ethyleneoxide) (PEO-like) film coated glass slides to study adhesion of stem cells. The arrays were generated by using a non-contact printing technology. The stability and the quality of the spots of fibronectin, used as protein model, were assessed by time of flight secondary ion mass spectrometry (ToF-SIMS), ellipsometry and atomic force microscopy (AFM). It was found that saturation with a mass density of 112 +/- 4 ng/cm2 is reached when protein solutions at concentrations higher than 84 mg/ml are spotted. Fibronectin on the surface form a uniform sub-monolayer with a surface coverage that depends on the spotting solution concentration, as qualitatively demonstrated by AFM measurements. The active conformation of the spotted fibronectin was verified by performing an immunoassay with antibodies specific for the fibronectin RGD sequence by surface plasmon resonance (SPR) imaging. An immunorecognition efficiency of up to 22% was found for a spot with 3% coverage as estimated by ellipsometry. Human umbilical cord blood neural stem cells (HUCB-NSCs) were cultured on different ECM proteins (fibronectin, laminin, collagen I, collagen III and collagen V) arrays and showed protein concentration dependent adhesion on the micro-spots. The cell nuclei were stained for cell counting and preliminary specific cell staining was performed to evaluate the differentiation stage of HUCB-NSCs on such spots. The array platform developed in this study provides a promising approach to investigate in high throughput manner how surfaces patterned with extracellular matrix (ECM) proteins influence stem cell adhesion and development.JRC.I.4-Nanobioscience

Vessel grouping patterns in subfamilies Apocynoideae and Periplocoideae confirm phylogenetic value of wood structure within Apocynaceae

This study contributes to our understanding of the phylogenetic significance and major evolutionary trends in the wood of the dogbane family (Apocynaceae), one of the largest and economically most important angiosperm families. Based on LM and SEM observations of 56 Apocynoideae species—representing all currently recognized tribes—and eight Periplocoideae, we found striking differences in vessel grouping patterns (radial multiples vs. large clusters) between the mainly nonclimbing apocynoid tribes (Wrightieae, Malouetieae, Nerieae) and the climbing lineages (remaining Apocynoideae and Periplocoideae). The presence of large vessel clusters in combination with fibers in the ground tissue characterizing the climbing Apocynoideae and Periplocoideae clearly contrasts with the climbing anatomy of the rauvolfioids (solitary vessels plus tracheids in ground tissue), supporting the view that (1) the climbing habit has evolved more than once in Apocynaceae, (2) the three nonclimbing apocynoid tribes are basal compared to the climbing apocynoids, and (3) Periplocoideae belong to the crown clade. The wood anatomy within the nonclimbing and climbing lineages is rather homogeneous, although a combination of specific characters (e.g. presence of septate fibers, axial parenchyma distribution, abundance of uniseriate compared to multiseriate rays, and presence and location of prismatic crystals) may be used to identify several tribes

Woodiness within the Spermacoceae–Knoxieae alliance (Rubiaceae): retention of the basal woody condition in Rubiaceae or recent innovation?

† Background and Aims The tribe Spermacoceae is essentially a herbaceous Rubiaceae lineage, except for some species that can be described as ‘woody’ herbs, small shrubs to treelets, or lianas. Its sister tribe Knoxieae contains a large number of herbaceous taxa, but the number of woody taxa is higher compared to Spermacoceae. The occurrence of herbaceous and woody species within the same group raises the question whether the woody taxa are derived from herbaceous taxa (i.e. secondary woodiness), or whether woodiness represents the ancestral state (i.e. primary woodiness). Microscopic observations of wood anatomy are combined with an independent molecular phylogeny to answer this question. † Methods Observations of wood anatomy of 21 woody Spermacoceae and eight woody Knoxieae species, most of them included in a multi-gene molecular phylogeny, are carried out using light microscopy. † Key Results Observations of wood anatomy in Spermacoceae support the molecular hypothesis that all the woody species examined are secondary derived. Well-known wood anatomical characters that demonstrate this shift from the herbaceous to the woody habit are the typically flat or decreasing length vs. age curves for vessel elements, the abundance of square and upright ray cells, or even the (near-) absence of rays. These socalled paedomorphic wood features are also present in the Knoxieae genera Otiophora, Otomeria, Pentas, Pentanisia and Phyllopentas. However, the wood structure of the other Knoxieae genera observed (Carphalea, Dirichletia and Triainolepis) is typical of primarily woody taxa. † Conclusions In Spermacoceae, secondary woodiness has evolved numerous times in strikingly different habitats. In Knoxieae, there is a general trend from primary woodiness towards herbaceousness and back to (secondary) woodiness

The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6

Ablation of the cellular prion protein PrPC leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrPC prevents the disease1, suggesting that PrPC acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrPC-deficient mice is reduced, suggesting that PrPC acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23–120) of PrPC triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen2. A KKRPKPG-containing PrPC-derived peptide (FT23–50) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT23–50 and the equivalent type-IV collagen peptide. We conclude that PrPC promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrPC, these observations are relevant to the pathogenesis of demyelinating polyneuropathies—common debilitating diseases for which there are limited therapeutic options