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

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

BDNF stimulation of protein synthesis in cortical neurons requires the map kinase-interacting kinase MNK1

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m7GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons

EXPERIMENTAL STUDIES OF MITIGATION MATERIALS FOR BLAST INDUCED TBI

The objective of this experimental study is to compare the effects of various materials obstructing the flow of a blast wave and the ability of the given material to reduce the damage caused by the blast. Several methods of energy transfer in blast wave flows are known or expected including: material interfaces with impedance mismatches, density changes in a given material, internal shearing, and particle fracture. The theory applied to this research is that the greatest energy transfer within the obstructing material will yield the greatest mitigation effects to the blast. Sample configurations of foam were varied to introduce material interfaces and filler materials with varying densities and impedances (liquids and powders). The samples were loaded according to a small scale blast produced by an explosive driven shock tube housing gram-range charges. The transmitted blast profiles were analyzed for variations in impulse characteristics and frequency components as compared to standard free field profiles. The results showed a rounding effect of the transmitted blast profile for all samples with the effects of the low density fillers surpassing all others tested.United States. Office of Naval Research (N00014-08-1-0261

Ornithine Decarboxylase mRNA is Stabilized in an mTORC1-dependent Manner in Ras-transformed Cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development

Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. Pathological myocardial growth is a risk factor for cardiac failure to which faster protein synthesis is a major driving element. We aimed to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are due to alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other non-dividing primary cells. To address this, an optimized QuaNCAT LC/MS method was used to selectively quantify newly synthesized proteins in such cells. The study showed both classical (phenylephrine; PE) and more recent (insulin) pathological cardiac hypertrophic agents increased the synthesis of proteins involved in glycolysis, the Krebs cycle / beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was upregulated by signaling through mTORC1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was upregulated in rat hearts following TAC. This isoform possesses specific regulatory properties that may be involved in metabolic remodeling and as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly, PKM2 and eEF1 were not up-regulated in pregnancy or exercise induced CH, suggesting them as pathological CH specific markers. The study methods may be of utility to the examination of protein synthesis in primary cells

The Role of Interleukin-1 and Interleukin-18 in Pro-Inflammatory and Anti-Viral Responses to Rhinovirus in Primary Bronchial Epithelial Cells

Human Rhinovirus (HRV) is associated with acute exacerbations of chronic respiratory disease. In healthy individuals, innate viral recognition pathways trigger release of molecules with direct anti-viral activities and pro-inflammatory mediators which recruit immune cells to support viral clearance. Interleukin-1alpha (IL-1α), interleukin-1beta (IL-1β) and interleukin-18 (IL-18) have critical roles in the establishment of neutrophilic inflammation, which is commonly seen in airways viral infection and thought to be detrimental in respiratory disease. We therefore investigated the roles of these molecules in HRV infection of primary human epithelial cells. We found that all three cytokines were released from infected epithelia. Release of these cytokines was not dependent on cell death, and only IL-1β and IL-18 release was dependent on caspase-1 catalytic activity. Blockade of IL-1 but not IL-18 signaling inhibited up-regulation of pro-inflammatory mediators and neutrophil chemoattractants but had no effect on virus induced production of interferons and interferon-inducible genes, measured at both mRNA and protein level. Similar level of virus mRNA was detected with and without IL-1RI blockade. Hence IL-1 signaling, potentially involving both IL-1β and IL-1α, downstream of viral recognition plays a key role in induction of pro-inflammatory signals and potentially in recruitment and activation of immune cells in response to viral infection instigated by the epithelial cells, whilst not participating in direct anti-viral responses