Evaluation of mTOR-regulated mRNA translation.

mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR such as rapamycin (which impairs some of its functions) or mTOR kinase inhibitors (which probably block all functions).To assess effects of mTOR inhibition on general protein synthesis in cells, the incorporation of radiolabelled amino acids into protein is measured. This does not yield information on the effects of mTOR on the synthesis of specific proteins. To do this, two methods are described. In one, stable-isotope labelled amino acids are used, and their incorporation into new proteins is determined using mass spectrometric methods. The proportions of labelled vs. unlabeled versions of each peptide from a given protein provide quantitative information about the rate of that protein's synthesis under different conditions. Actively translated mRNAs are associated with ribosomes in polyribosomes (polysomes); thus, examining which mRNAs are found in polysomes under different conditions provides information on the translation of specific mRNAs under different conditions. A method for the separation of polysomes from non-polysomal mRNAs is describe

Birefringence measurements in single crystal sapphire and calcite shocked along the a axis

Calcite and sapphire were shock compressed along the ⟨101⎯⎯0⟩ direction (a axis) in a plate impact configuration. Polarimetery and Photonic Doppler Velocimetery (PDV) were used to measure the change in birefringence with particle velocity in the shock direction. Results for sapphire agree well with linear photoelastic theory and current literature showing a linear relationship between birefringence and particle velocity up to 310 m s−1. A maximum change in birefringence of 5% was observed. Calcite however showed anomolous behaviour with no detectable change in birefringence (less than 0.1%) over the range of particle velocities studied (up to 75 m s−1)

Shockwaves in converging geometries

Plate impact experiments are a powerful tool in equation of state (EOS) development, but are inherently limited by the range of impact velocities accessible to the gun. In an effort to dramatically increase the range of pressures which can be studied with available impact velocities, a new experimental technique is being developed. The possibility of using a confined converging target to focus Shockwaves and produce a large amplitude pressure pulse is examined. When the planar shock resulting from impact enters the converging target the impedance mismatch at the boundary of the confinement produces reflected Mach waves and the subsequent wave interactions produce a diffraction cycle resulting in increases in the shock strength with each cycle. Since this configuration is limited to relatively low impedance targets, a second technique is proposed in which the target is two concentric cylinders designed such that the inner cylinder will have a lower shock velocity than the much larger shock velocity in the outer cylinder. The resulting dispersion in the wave front creates converging shocks, which will interact and eventually result in a steady Mach configuration with an increase in pressure in the Mach disk. Numerical simulations indicate a significant increase in pressure for both methods and show promise for the proposed concepts

Advances in Shock Compression of Mantle Materials and Implications

Hugoniots of lower mantle mineral compositions are sensitive to the conditions where they cross phase boundaries including both polymorphic phase transitions and partial to complete melting. For SiO_2, the Hugoniot of fused silica passes from stishovite to partial melt (73 GPa, 4600 K) whereas the Hugoniot of crystal quartz passes from CaCi_2 structure to partial melt (116 GPa, 4900 K). For Mg_2SiO_4, the forsterite Hugoniot passes from the periclase +MgSiO_3 (perovskite) assemblage to melt before 152 GPa and 4300 K, whereas the wadsleyite Hugoniot transforms first to periclase +MgSiO_3 (post-perovskite) and then melts at 151 GPa and 4160 K. Shock states achieved from crystal enstatite are molten above 160 GPa. High-pressure Grüneisen parameters for molten states of MgSiO_3 and Mg_2SiO_4 increase markedly with compression, going from 0.5 to 1.6 over the 0 to 135 GPa range. This gives rise to a very large (>2000 K) isentropic rise in temperature with depth in thermal models of a primordial deep magma ocean within the Earth. These magma ocean isentropes lead to models that have crystallization initiating at mid-lower mantle depths. Such models are consistent with the suggestion that the present ultra-low velocity zones, at the base of the lowermost mantle, represent a dynamically stable, partially molten remnant of the primordial magma ocean. The new shock melting data for silicates support a model of the primordial magma ocean that is concordant with the Berkeley-Caltech iron core model [1] for the temperature at the center of the Earth

Shock temperatures of preheated MgO

Shock temperature measurements via optical pyrometry are being conducted on single-crystal MgO preheated before compression to 1905–1924 K. Planar shocks were generated by impacting hot Mo(driver plate)-MgO targets with Mo or Ta flyers launched by the Caltech two-stage light-gas gun up to 6.6 km/s. Quasi-brightness temperature was measured with 2–3% uncertainty by a 6-channel optical pyrometer with 3 ns time resolution, over 500–900 nm spectral range. A high-power, coiled irradiance standard lamp was adopted for spectral radiance calibration accurate to 5%. In our experiments, shock pressure in MgO ranged from 102 to 203 GPa and the corresponding temperature varied from 3.78 to 6.53 kK. For the same particle velocity, preheated MgO Hugoniot has about 3% lower shock velocity than the room temperature Hugoniot. Although model shock temperatures calculated for the solid phase exceeded our measurements by ~5 times the uncertainty, there was no clear evidence of MgO melting, up to the highest compression achieved

Replicating landmine blast loading in cellular <i>in Vitro</i> models

Trauma arising from landmines and improvised explosive devices promotes heterotopic ossification, the formation of extra-skeletal bone in non-osseous tissue. To date, experimental platforms that can replicate the loading parameter space relevant to improvised explosive device and landmine blast wave exposure have not been available to study the effects of such non-physiological mechanical loading on cells. Here, we present the design and calibration of three distinct in vitro experimental loading platforms that allow us to replicate the spectrum of loading conditions recorded in near-field blast wave exposure. We subjected cells in suspension or in a three-dimensional hydrogel to strain rates up to 6000 s-1and pressure levels up to 45 MPa. Our results highlight that cellular activation is regulated in a non-linear fashion - not by a single mechanical parameter, it is the combined action of the applied mechanical pressure, rate of loading and loading impulse, along with the extracellular environment used to convey the pressure waves. Finally, our research indicates that PO MSCs are finely tuned to respond to mechanical stimuli that fall within defined ranges of loading

Polymer Nanocomposite Materials Based on Carbon Nanotubes

(Invited Book Chapter

Hegel, Plotinus, and the Problem of Evil

The article begins by defining evil, rejecting proffered negative definitions, for example, evil is privation, from which little further insight is obtainable, nor progress to be made towards a resolution of the problem, in favor of a more a constructive definition of evil in terms of an abnegation of responsibility for, or commitment to, one’s dictions or actions. This is an attitude that is both irrational and unintelligible, enabling a connection to be made between Plotinus’ view of evil as unintelligible matter, that is, impassive and inactive nature; and Hegel’s view of evil as that which is real and active, but against reason. For Plotinus, evil is formless matter, matter that is unintelligible, separated from the intellect, unlimited because not bounded by conceptual categories; and hence, what is evil is all darkness. For Hegel, for whom the rational is actual and the actual is rational, evil is an irrational perversion of the world’s inner essential rationality. But these ideas can be connected, for the purpose of finding a solution to the problem of evil, through a more specific understanding of the concept that we offer; that evil is a failure of commitment, a denial of responsibility for one’s actions; and this is in itself both irrational and unintelligible. We can then demonstrate that what that evil, though it certainly exists, is imperfection, and the good, being rational, is already complete and fulfilled and firmly established in the world, and therefore evil cannot possibly oppose it from any position of equivalence; for the good is rational and therefore true, whereas evil is irrational and therefore false

Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission