Extracellular Matrix Mineralization Promotes E11/gp38 Glycoprotein Expression and Drives Osteocytic Differentiation

Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy (15N, 13C, 2H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for “promoting motions” in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate

Syndromic surveillance to assess the potential public health impact of the Icelandic volcanic ash plume across the United Kingdom, April 2010

The Eyjafjallajökull volcano in Iceland erupted on 14 April 2010 emitting a volcanic ash plume that spread across the United Kingdom and mainland Europe. The Health Protection Agency and Health Protection Scotland used existing syndromic surveillance systems to monitor community health during the incident: there were no particularly unusual increases in any of the monitored conditions. This incident has again demonstrated the use of syndromic surveillance systems for monitoring community health in real time

Gravitational-wave astronomy: the high-frequency window

This contribution is divided in two parts. The first part provides a text-book level introduction to gravitational radiation. The key concepts required for a discussion of gravitational-wave physics are introduced. In particular, the quadrupole formula is applied to the anticipated ``bread-and-butter'' source for detectors like LIGO, GEO600, EGO and TAMA300: inspiralling compact binaries. The second part provides a brief review of high frequency gravitational waves. In the frequency range above (say) 100Hz, gravitational collapse, rotational instabilities and oscillations of the remnant compact objects are potentially important sources of gravitational waves. Significant and unique information concerning the various stages of collapse, the evolution of protoneutron stars and the details of the supranuclear equation of state of such objects can be drawn from careful study of the gravitational-wave signal. As the amount of exciting physics one may be able to study via the detections of gravitational waves from these sources is truly inspiring, there is strong motivation for the development of future generations of ground based detectors sensitive in the range from hundreds of Hz to several kHz.Comment: 21 pages, 5 figures, Lectures presented at the 2nd Aegean Summer School on the Early Universe, Syros, Greece, September 200

Thermal Adaptation of Dihydrofolate Reductase from the Moderate ThermophileGeobacillus stearothermophilus

The thermal melting temperature of dihydrofolate reductase from Geobacillus stearothermophilus (BsDHFR) is 30 °C higher than that of its homologue from the psychrophile Moritella profunda. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of G. stearothermophilus, which is characterized by temperatures that can reach 75 °C

Forecast climate change impact on pore-water pressure regimes for the design and assessment of clay earthworks

Understanding and mitigating the impact of climate change on the built environment is becoming increasingly important worldwide. Earthworks (embankments and cuttings) supporting road and rail transportation networks often have direct contact with the atmosphere and are therefore influenced by extreme weather events and seasonal weather patterns. Atmospheric wetting and drying alters pore-water pressures (PWP) within earthworks, potentially contributing to the deformation and failure of earthwork slopes. Consequently, it is essential to understand the influence of climate change on PWPs within earthwork slopes, to inform strategies for their design, assessment and maintenance. Extensive one-dimensional seepage analyses were carried out for typical railway embankments in the London area. The analyses showed that forecast hotter, drier summers will increase the water storage capacity of earthworks. This will lead to increased net infiltration in the winter months due to both a forecast increase in rainfall, and a longer time being required to saturate the soil pores and bring the water table back to the slope surface. Hence, despite the forecast increase in winter rainfall, this will not lead to higher design PWP regimes. The analyses were conducted for the London area, but the methodology and conceptual framework can be readily adapted for other locations