Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2) affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo

The quasi-quantum treatment of rotationally inelastic scattering from a hard shell potential: its derivation and practical use

The QQT is a quasi-quantum mechanical treatment of the collision between molecules. Instead of a partial wave expansion approach, it uses a kind of Feynman path integral method that exploits the path length differences originating from the different orientations of an anisotropic molecule. As a result, the QQT provides valuable physical insight while requiring very little computational effort. The current paper gives a systematic derivation of the QQT and explains its underlying principles. The expression for the scattering amplitude is shown to be self-consistent, without any normalisation factors, when the rotational energy level spacing is negligible. The constant curvature approximation that is presented makes the QQT conceptually even more simple, and its effect on the calculated differential cross-sections (DCSs) turns out to be small. As examples we present QQT calculations of the DCSs for Ne-CO(1) and He-NO(2), at collision energies of, respectively, 511 cm-1 and 514 cm-1. The anisotropy of the hard shell potential energy surface for Ne-CO in terms of the incoming de Broglie wavelength is about twice as large as for He-NO. This leads to state-to-state DCSs that have up to three maxima of comparable amplitude, instead of only one large maximum as is found for He-NO. The QQT results for these two applications are compared with results from close coupling calculations

Numerical calculation of permeability of periodic porous materials: application to periodic arrays of spheres and 3D scaffold microstructures

In this paper, an efficient numerical method is proposed to calculate the anisotropic permeability in porous materials characterized by a periodic microstructure. This method is based on pore‐scale fluid dynamic simulations using a static volume of fluid method. Unlike standard solution procedures for this type of problem, we here solve an average constitutive equation over both fluid and solid domain by use of a subgrid model to accurately capture momentum transfer from the fluid to solid interface regions. Using numerical simulations on periodic arrays of spheres, we first demonstrate that, by using the subgrid interface model, more accurate results can be produced, for the velocity and pressure fields, than via more conventional approaches. We then apply numerical upscaling over the unit cell to calculate the full anisotropic permeability from the pore‐scale numerical results. The obtained permeability values for a variety of periodic arrays of spheres in different arrangements and packing orders are in good agreement with semianalytical results reported in literature. This validation allows for the permeability assessment of more complex structures such as isotropic gyroid structures, or anisotropic cases, here modeled in their simplest form, the ellipsoidal inclusion