In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.     

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.     

ANOXIA AND BRIGHTNESS DISCRIMINATION

1. Brightness discrimination has been studied with individuals breathing oxygen concentrations corresponding to 7 altitudes between sea level and 17,000 feet. The brightnesses were 0.1, 0.01, and 0.001 millilambert involving only daylight (cone) vision. 2. At these light intensities, brightness discrimination begins to deteriorate at fairly low altitudes. The deterioration is obvious at 8,000 feet, and becomes marked at 15,000 feet, where at low brightness, the contrast must be increased 100 per cent over the sea level value before it can be recognized. 3. The impairment of brightness discrimination with increase in altitude is greater at higher altitudes than at lower. The impairment starts slowly and becomes increasingly rapid the higher the altitude. 4. Impairment of brightness discrimination varies inversely with the light intensity. It is most evident under the lowest light intensities studied, but shows in all of them. However, it decreases in such a way that the deterioration is negligible in full daylight and sunlight. 5. The thresholds of night (rod) vision and day (cone) vision are equally affected by anoxia. 6. The quantitative form of the relation between brightness discrimination ΔI/I and the prevailing brightness I remains the same at all oxygen concentrations. The curve merely shifts along the log I axis, and the extent of the shift indicates the visual deterioration. 7. The data are described in terms of retinal chemistry. Since anoxia causes only a shift in log I it is shown that the photochemical receptor system cannot be affected. Instead the conversion of photochemical change into visual function is impaired in such a way that the conversion factor varies as the fourth power of the arterial oxygen saturation.     

A Bayesian calibration model for combining different pre-processing methods in Affymetrix chips

Background  

In gene expression studies a key role is played by the so called "pre-processing", a series of steps designed to extract the signal and account for the sources of variability due to the technology used rather than to biological differences between the RNA samples. At the moment there is no commonly agreed gold standard pre-processing method and each researcher has the responsibility to choose one method, incurring the risk of false positive and false negative features arising from the particular method chosen.     

Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome

MicroRNAs usually interact with 3' noncoding regions (3'NCRs) of target mRNAs leading to downregulation of mRNA expression. In contrast, liver-specific microRNA miR-122 interacts with the 5' end of the hepatitis C virus RNA genome, resulting in increased viral RNA abundance. We find that inserting the viral miR-122 binding site into the 3' noncoding region of a reporter mRNA leads to downregulation of mRNA expression, indicating that the location of the miR-122 binding site dictates its effect on gene regulation. Furthermore, we discovered an adjacent, second miR-122 binding site, separated from the first by a highly conserved 14-nucleotide sequence. Mutational analysis demonstrates that both miR-122 binding sites in a single viral genome are occupied by the microRNA and function cooperatively to regulate target gene expression. These findings set a paradigm for dual, position-dependent functions of tandem microRNA-binding sites. Targeting an oligomeric microRNA complex offers potential as an antiviral-intervention strategy.     

Two distinct P element subfamilies in the genome of Drosophila bifasciata

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5 end and 14 by at the 3 end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.