Determination of Effector Binding Affinities Using PAC

Determination of binding affinity and thermodynamic parameters of interactions between two molecules is a common procedure in biochemical contexts. Steady-state fluorescence titration is one of the most commonly used techniques to determine the binding affinity of small effector compounds and ligands to large biomolecules such as proteins. This method, however requires 1) the ligand to be fluorescent and exhibit a measureable change in fluorescence intensity between the bound and free states or 2) a fluorescent residue (e.g. tryptophan) near the ligand binding site whose fluorescence changes as a result of ligand binding. The new technique presented here utilizes photoacoustic calorimetry (PAC) in conjunction with conventional titration techniques to determine the binding affinity and thermodynamic parameters of a ligand binding to a protein. This technique does not require a fluorescent ligand or amino acid residue to be present and instead utilizes the change in the PAC signal as a function of ligand concentration to determine these parameters. So far, we have used this technique to determine the dissociation constant (KD) and enthalpy change (?H) for binding of inositol hexakisphosphate (IHP) (KD = 73.5 mM at 20 °C, ?H = 9.8 kJ mol-1) to human adult hemoglobin (HbA). Future trials will look at other common ligands for HbA as well as other heme proteins. Being able to determine the binding affinity and thermodynamic parameters for ligand binding to proteins is extremely useful for understanding the environment of the ligand binding site potentially allowing us to create new and more effective drugs. The use of PAC may provide a possible method of evaluating these characteristics quickly

The Stargazin-Related Protein {gamma}7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including CaV2.2

The role(s) of the novel stargazin-like {gamma}-subunit proteins remain controversial. We have shown previously that the neuron-specific {gamma}7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of {gamma}7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of {gamma}7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous {gamma}7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed {gamma}7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of {gamma}7 is essential for all its effects, and we show that {gamma}7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa, and this enhancement is prevented by a concentration of {gamma}7 that alone has no effect on IBa. The effect of {gamma}7 is selective for certain mRNAs because it had no effect on {alpha}2{delta}-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that {gamma}7 plays a role in stabilizing CaV2.2 mRNA

An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype

Strong stability preserving explicit Runge-Kutta methods of maximal effective order

We apply the concept of effective order to strong stability preserving (SSP) explicit Runge-Kutta methods. Relative to classical Runge-Kutta methods, methods with an effective order of accuracy are designed to satisfy a relaxed set of order conditions, but yield higher order accuracy when composed with special starting and stopping methods. We show that this allows the construction of four-stage SSP methods with effective order four (such methods cannot have classical order four). However, we also prove that effective order five methods - like classical order five methods - require the use of non-positive weights and so cannot be SSP. By numerical optimization, we construct explicit SSP Runge-Kutta methods up to effective order four and establish the optimality of many of them. Numerical experiments demonstrate the validity of these methods in practice.Comment: 17 pages, 3 figures, 8 table

Optically induced forces and torques:Interactions between nanoparticles in a laser beam

Distinctive optical forces and torques arise between nanoparticles irradiated by intense laser radiation. These forces, associated with a pairwise process of stimulated scattering, prove to enable the possibility of producing significant modifications to both the form and magnitude of interparticle forces, with additional contributions arising in the case of dipolar materials. Moreover, such forces have the capacity to generate unusual patterns of nanoscale response, entirely controlled by the input beam characteristics- principally the optical frequency, intensity, and polarization. Based on quantum electrodynamical theory, a general result is secured for the laser-induced force under arbitrary conditions, incorporating both static and dynamic coupling mechanisms. Specific features of the results are identified for pairs of particles with prolate cylindrical symmetry, e.g., carbon nanotubes, where it is shown that the laser-induced forces and torques are sensitive functions of the pair spacing and orientation, and the laser beam geometry; significantly, they can be either repulsive or attractive according to conditions. For nanoparticles trapped in a Laguerre-Gaussian laser beam the results also reveal additional and highly distinctive torques that suggest further possibilities for nanomanipulation with light. The paper concludes with a discussion on several potential applications of such forces. © 2005 The American Physical Society

Deficiency in β1,3-Galactosyltransferase of a Leishmania major Lipophosphoglycan Mutant Adversely Influences the Leishmania-Sand Fly Interaction

To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi

Failure parameter identification and validation for a dual-phase 780 steel sheet

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.ijsolstr.2017.06.018 © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/A hybrid experimental-numerical procedure was implemented to determine the failure surface of a dual-phase 780 steel sheet as a function of the effective plastic strain, triaxiality, and Lode parameter using butterfly specimens with in situ digital image correlation strain measurement and supporting finite element calculations. A butterfly-type test specimen was employed to experimentally obtain stress states ranging from simple shear to plane strain tension including mixed tensile and shear loading. The numerically-derived failure surface was implemented into the phenomenological GISSMO damage model in the commercial finite element code LS-DYNA and the accuracy of the failure surface was determined using finite element predictions of the characterization experiments. A series of independent validation experiments related to sheet metal forming were performed including a hole tension test, a conical and flat punch hole expansion test, and a hemispherical punch test. The finite element models utilizing the damage model were able to accurately reproduce the load–displacement and surface strains of the sheet material for both the characterization and validation experiments. Prediction of the failure orientation and location compared favorably to each of the validation tests

Urban policy

The employment relationship – that between employer and employee – is at the heart of capitalism and a core issue for public policy. Governments create rules, policies and institutions within which employees, their representatives, employers and their representatives, operate. The interest to governments when creating policy includes the form that bargaining takes, wage and employment levels, the nature and effects of contracting and the rights of workers – much of this boiling down to issues of power. In recent decades, major policy issues have included the federal Labor government’s Prices and Incomes Accords in the 1980s and 1990s, the Coalition government’s ‘WorkChoices’ legislation, the shift to enterprise bargaining, and developments in such areas as minimum wages and pay equity. In this chapter we outline the matters at stake, the players, the policy processes and some of the key issues