Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro

<p>Abstract</p> <p>Background</p> <p>The interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells <it>in vitro </it>in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface.</p> <p>Results</p> <p>A protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation <it>in vitro </it>over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis.</p> <p>Conclusion</p> <p>These data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.</p

Hepatic effects of tartrazine (E 102) after systemic exposure are independent of oestrogen receptor interactions in the mouse

Tartrazine is a food colour that activates the transcriptional function of the human oestrogen receptor alpha in an in vitro cell model. Since oestrogens are cholestatic, we hypothesised tartrazine will cause periportal injury to the liver in vivo. To test this hypothesis, tartrazine was initially administered systemically to mice resulting in a periportal recruitment of inflammatory cells, increased serum alkaline phosphatase activity and mild periportal fibrosis. To determine whether an oestrogenic effect may be a key event in this response, tartrazine, sulphonated metabolites and a food additive contaminant were screened for their ability to interact with murine oestrogen receptors. In all cases, there were no interactions as agonists or antagonists and further, no oestrogenicity was observed with tartrazine in an in vivo uterine growth assay. To examine the relevance of the hepatic effects of tartrazine to its use as a food additive, tartrazine was orally administered to transgenic NF-κB-Luc mice. Pre- and concurrent oral treatment with alcohol was incorporated given its potential to promote gut permeability and hepatic inflammation. Tartrazine alone induced NF- κB activities in the colon and liver but there was no periportal recruitment of inflammatory cells or fibrosis. Tartrazine, its sulphonated metabolites and the contaminant inhibited sulphotransferase activities in murine hepatic S9 extracts. Given the role of sulfotransferases in bile acid excretion, the initiating event giving rise to periportal inflammation and subsequent hepatic pathology through systemic tartrazine exposure is therefore potentially associated an inhibition of bile acid sulphation and excretion and not on oestrogen receptor-mediated transcriptional function. However, these effects were restricted to systemic exposures to tartrazine and did not occur to any significant effect after oral exposure

Impedimetric array in polymer microfluidic cartridge for low cost point-of-care diagnostics

International audienc

Sorghum Genome Sequencing by Methylation Filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis

Adaptation of a Point-of-Care Canine Progesterone Test for Use of Parturition Prediction in Captive Asian Elephants (Elephas Maximus): Proof of Concept

In the Asian elephant (Elephas maximus), the levels of progesterone products 5α-pregnane, 3α-hydroxypregnane, and 17α-progesterone are elevated during pregnancy. Detection of a sudden decrease in blood progesterone product levels in the final days of pregnancy is considered an objective way of predicting impending parturition. Point-of-care (POC) tests eliminate the cost involved in transporting samples to an external laboratory and provide an almost instant result, facilitating decision-making for animal monitoring and management. This proof-of-concept study aims to investigate the ability of the AgPlus POC immunoassay system to measure 4-pregnen-3,20-dione in pregnant elephant serum samples and adapt the method for detection of the preparturient progesterone decrease. Frozen serum samples of two pregnant elephants (N = 82) and fresh serum samples of one pregnant elephant (N = 10) were analyzed using both the POC method and a radioimmunoassay in a reference laboratory. Statistical analysis of the data showed that there was no significant difference between the two methods for detection of the progesterone drop, indicating that the POC method can be considered appropriate for use in elephant parturition prediction. Refinement of the methodology, an increase of sample size, and temporal tandem radioimmunoassay would be required to further validate this method for use in elephant reproductive management

A molecular picture of surface interactions of organic compounds on prevalent indoor surfaces: limonene adsorption on SiO2.

Indoor surfaces are often coated with organic compounds yet a molecular understanding of what drives these interactions is poorly understood. Herein, the adsorption and desorption of limonene, an organic compound found in indoor environments, on hydroxylated silica (SiO2) surfaces, used to mimic indoor glass surfaces, is investigated by combining vibrational spectroscopy, atomistic computer simulations and kinetic modeling. Infrared spectroscopy shows the interaction involves hydrogen-bonding between limonene and surface O-H groups. Atomistic molecular dynamics (MD) simulations confirm the existence of π-hydrogen bonding interactions, with one or two hydrogen bonds between the silica O-H groups and the carbon-carbon double bonds, roughly one third of the time. The concentration and temperature dependent adsorption/desorption kinetics as measured by infrared spectroscopy were reproduced with a kinetic model, yielding the adsorption enthalpy of ∼55 kJ mol-1, which is consistent with the value derived from the MD simulations. Importantly, this integrated experimental, theoretical and kinetic modeling study constitutes a conceptual framework for understanding the interaction of organic compounds with indoor relevant surfaces and thus provides important insights into our understanding of indoor air chemistry and indoor air quality

Differential methylation of genes and repeats in land plants

The hypomethylated fraction of plant genomes is usually enriched in genes and can be selectively cloned using methylation filtration (MF). Therefore, MF has been used as a gene enrichment technology in sorghum and maize, where gene enrichment was proportional to genome size. Here we apply MF to a broad variety of plant species spanning a wide range of genome sizes. Differential methylation of genic and non-genic sequences was observed in all species tested, from non-vascular to vascular plants, but in some cases, such as wheat and pine, a lower than expected level of enrichment was observed. Remarkably, hexaploid wheat and pine show a dramatically large number of gene-like sequences relative to other plants. In hexaploid wheat, this apparent excess of genes may reflect an abundance of methylated pseudogenes, which may thus be more prevalent in recent polyploids

Silicon Surface Nanostructuring for Covalent Immobilization of Biomolecules

We present a straightforward strategy to control the average distance of immobilized biomolecules on silicon surfaces. We exploit the reaction taking place between the amino residues within the biomolecules (lysine groups of proteins or the N-terminus of oligomers of peptide nucleic acid, PNA) and the aldehyde-terminated groups presented in a mixed aldehyde/alkyl organic monolayer on a silicon surface. The mixed monolayers were prepared by a thermal reaction of hydrogen-terminated Si(111) with a mixture of undecene and undecenyl-aldehyde. We quantitatively evaluate the surface concentration of aldehyde in the monolayer by atomic force microscopy and an intensity analysis of core level X-ray photoemission spectroscopy peaks. These complementary techniques show that the surface density of the reactive terminal groups reflects the mole fraction of aldehyde in the reactive solution used to modify the silicon surface. The further immobilization of proteins or peptide nucleic acids on the monolayer shows that the density of biomolecules reproduces the aldehyde surface density, which indicates a specific covalent attachment and a negligible nonspecific adsorption. The proposed procedure makes possible to control the average distance of the immobilized active biomolecules on the silicon surface, which could be of great relevance for applications in the interdisciplinary field of biosensors.Peer reviewe