Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH3, 2′-H or 2′-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH3 suppressed processing at the major aberrant m−1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2′-OCH3 supports a model where the extra methylene (LNA) or methyl group (2′-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50 μm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors

2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), affect millions of people every year and so far, there are no therapeutic cures available. Even though animal and histological models have been of great aid in understanding disease mechanisms and identifying possible therapeutic strategies, in order to find disease-modifying solutions there is still a critical need for systems that can provide more predictive and physiologically relevant results. One possible avenue is the development of patient-derived models, e.g. by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), which can then be differentiated into any cell type for modelling. These systems contain key genetic information from the donors, and therefore have enormous potential as tools in the investigation of pathological mechanisms underlying disease phenotype, and progression, as well as in drug testing platforms. hiPSCs have been widely cultured in 2D systems, but in order to mimic human brain complexity, 3D models have been proposed as a more advanced alternative. This review will focus on the use of patient-derived hiPSCs to model AD, PD, HD and ALS. In brief, we will cover the available stem cells, types of 2D and 3D culture systems, existing models for neurodegenerative diseases, obstacles to model these diseases in vitro, and current perspectives in the field

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly-L-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2.

Contains fulltext : 88529.pdf (publisher's version ) (Closed access)In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (muHA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is unknown. To examine this, rat bone marrow (RBM) cells were cultured on porous poly-L-lactic acid and composite scaffolds, with or without added BMP-2. Cell proliferation and differentiation were studied using DNA, alkaline phosphatase and scanning electron microscopic analysis. Further, genetic profiles were examined by microarray investigation. Results showed that the composite scaffold had no significant effect on the proliferation of RBM cells, but indicated a negative effect on cell differentiation. The addition of BMP-2 also had no significant effect on the proliferation of RBM cells, but differentiation towards the osteogenic lineage was confirmed. In the arrays results, the addition of BMP-2 alone led to the expression of genes involved in (minor) inflammation. The composite scaffold, and even more distinctly the combination of the composite scaffold with BMP-2, led to the expression of genes, based on gene ontology, connected to tumorigenesis. Therefore, CNT- and muHA-containing composite materials are not recommended as a bone restorative material.1 november 201

Utilisation of chemically stabilized arsenic-contaminated soil in a landfill cover

The aim of the study was to determine if an As-contaminated soil, stabilized using zerovalent iron (Fe0) and its combination with gypsum waste, coal fly ash, peat, or sewage sludge, could be used as a construction material at the top layer of the landfill cover. A reproduction of 2 m thick protection/vegetation layer of a landfill cover using a column setup was used to determine the ability of the amendments to reduce As solubility and stimulate soil functionality along the soil profile. Soil amendment with Fe0 was highly efficient in reducing As in soil porewater reaching 99 % reduction, but only at the soil surface. In the deeper soil layers (below 0.5 m), the Fe treatment had a reverse effect, As solubility increased dramatically exceeding that of the untreated soil or any other treatment by one to two orders of magnitude. A slight bioluminescence inhibition of Vibrio fischeri was detected in the Fe0 treatment. Soil amendment with iron and peat showed no toxicity to bacteria and was the most efficient in reducing dissolved As in soil porewater throughout the 2 m soil profile followed by iron and gypsum treatment, most likely resulting from a low soil density and a good air diffusion to the soil. The least suitable combination of soil amendments for As immobilization was a mixture of iron with coal fly ash. An increase in all measured enzyme activities was observed in all treatments, particularly those receiving organic matter. For As to be stable in soil, a combination of amendments that can keep the soil porous and ensure the air diffusion through the entire soil layer of the landfill cover is required

Microstructural characterisation of plasma sprayed thermal barrier coatings by quantitative image analysis

Communication to : Quantitative microscopy of high temperature materials conference, Sheffield (Great-Britain), November 22-24, 1999Available from INIST (FR), Document Supply Service, under shelf-number : 22419, issue : a.2000 n.89 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc