Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory

Ecology: a prerequisite for malaria elimination and eradication

* Existing front-line vector control measures, such as insecticide-treated nets and residual sprays, cannot break the transmission cycle of Plasmodium falciparum in the most intensely endemic parts of Africa and the Pacific * The goal of malaria eradication will require urgent strategic investment into understanding the ecology and evolution of the mosquito vectors that transmit malaria * Priority areas will include understanding aspects of the mosquito life cycle beyond the blood feeding processes which directly mediate malaria transmission * Global commitment to malaria eradication necessitates a corresponding long-term commitment to vector ecolog

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

High-resolution 3D analysis of mouse small-intestinal stroma.

Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation. In contrast to alternative current techniques, our protocol enables the entire villus to be visualized with increased spatial resolution of cell location, morphology and cell-cell interactions, thus allowing for easy quantification of phenotypes. The technique, which takes 7 d from mouse dissection to microscopic examination, will be useful for researchers who are interested in most aspects of intestinal biology, including mucosal immunology, infection, nutrition, cancer biology and intestinal microbiota

Current tidal power technologies and their suitability for applications in coastal and marine areas

A considerable body of research is currently being performed to quantify available tidal energy resources and to develop efficient devices with which to harness them. This work is naturally focussed on maximising power generation from the most promising sites, and a review of the literature suggests that the potential for smaller scale, local tidal power generation from shallow near-shore sites has not yet been investigated. If such generation is feasible, it could have the potential to provide sustainable electricity for nearby coastal homes and communities as part of a distributed generation strategy, and would benefit from easier installation and maintenance, lower cabling and infrastructure requirements and reduced capital costs when compared with larger scale projects. This article reviews tidal barrages and lagoons, tidal turbines, oscillating hydrofoils and tidal kites to assess their suitability for small-scale electricity generation in shallow waters. This is achieved by discussing the power density, scalability, durability, maintainability, economic potential and environmental impacts of each concept. The performance of each technology in each criterion is scored against axial-flow turbines, allowing for them to be ranked according to their overall suitability. The review suggests that tidal kites and range devices are not suitable for small-scale shallow water applications due to depth and size requirements respectively. Cross-flow turbines appear to be the most suitable technology, as they have high power densities and a maximum size that is not constrained by water depth

Broadband Monitoring Tracing the Evolution of the Jet and Disk in the Black Hole Candidate X-ray Binary MAXI J1659-152

MAXI J1659−152 was discovered on 2010 September 25 as a new X-ray transient, initially identified as a gamma-ray burst, but was later shown to be a new X-ray binary with a blackhole as the most likely compact object. Dips in the X-ray light curves have revealed that MAXI J1659−152 is the shortest period black hole candidate identified to date. Here we present the results of a large observing campaign at radio, submillimetre, near-infrared (nIR), optical and ultraviolet (UV) wavelengths. We have combined this very rich data set with the available X-ray observations to compile a broad-band picture of the evolution of this outburst. We have performed broad-band spectral modelling, demonstrating the presence of a spectral break at radio frequencies and a relationship between the radio spectrum and X-ray states. Also, we have determined physical parameters of the accretion disc and put them into context with respect to the other parameters of the binary system. Finally, we have investigated the radio–X-ray and nIR/optical/UV–X-ray correlations up to ∼3 yr after the outburst onset to examine the link between the jet and the accretion disc, and found that there is no significant jet contribution to the nIR emission when the source is in the soft or intermediate X-ray spectral state, consistent with our detection of the jet break at radio frequencies during these states

Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor

Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems use stirred cuvettes, stopped-flow apparatus, microfluidic systems, or other small sample containers. These methods may prove to be problematic if reactants or products adsorb to or react with the container’s surface. As an alternative approach, we have developed an acoustically-levitated drop reactor eventually intended to study enzyme-catalyzed reaction kinetics related to free radical and oxidative stress chemistry. Microliter-scale droplet generation, reactant introduction, maintenance, and fluid removal are all important aspects in conducting reactions in a levitated drop. A three capillary bundle system has been developed to address these needs. We report kinetic measurements for both luminol chemiluminescence and the reaction of pyruvate with nicotinamide adenine dinucleotide, catalyzed by lactate dehydrogenase, to demonstrate the feasibility of using a levitated drop in conjunction with the developed capillary sample handling system as a microreactor

A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

Extracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2(S897)). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.We find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2(S897), lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2(S897) in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM