183 research outputs found
Contractors’ Perspectives on Airfield and Highway Hot Mix Asphalt Pavement Projects
No full textThesis (Master's)--University of Washington, 2020Asphalt pavement covers most highways and airfields in the United States. While paving primarily occurs on highways, airfield projects carry greater risks for contractors and involve different management styles, construction execution methods, and project specifications. Failure to consider and quantify project differences may cause delays and financial repercussions. Therefore, this research answers the question, “From a paving contractor’s perspective, what are the differences between airfield and highway paving projects within the State of Washington?” Following a review of specifications and best practices guides, interviews with three Washington asphalt contractors yielded comparisons of project experiences with the Washington State Department of Transportation (WSDOT), the Federal Aviation Administration (FAA), and the Department of Defense (DoD). Research results may benefit general audiences and, more specifically, United States Air Force (USAF) pavement engineers. Sharing contractors’ perspectives about asphalt paving enlightens government representatives about project partners’ issues and encourages communication and awareness
Membrane transport modulates the pH-regulated feedback of an enzyme reaction confined within lipid vesicles
No full textUnderstanding ion transport dynamics in reactive vesicles is pivotal for exploring biological and chemical processes and essential for designing synthetic cells. In this work, we investigate how proton transport and membrane potential regulate pH dynamics in an autocatalytic enzyme reaction within lipid vesicles. Combining experimental and numerical methods, we demonstrate that compartmentalization within lipid membranes accelerates internal reactions, attributed to protection from the external acidic environment. In experiments, we explored how proton movement significantly impacts internal reactions by changing bilayer thickness, adding ion transporters, and varying buffers. Numerical investigations incorporated electrical membrane potential and capacitance into a kinetic model of the process, elucidating the mechanisms that dictate the control of reaction time observed in the experiment, driven by both electrical and chemical potential gradients. These findings establish a framework for controlling pH clock reactions via membrane changes and targeted manipulation of proton movement, which could aid in the design of synthetic cells with precise, controlled functionalities
Everyone needs good neighbours – the intricate relationship between the acetylcholine-receptor channel and its membrane environment
No full textCryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states
No full textComplex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia–reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the ‘active’ state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the ‘deactive’ state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I
Structural studies of enoyl acyl carrier protein reductase from Plasmodium falciparum and Toxoplasma gondii.
No full textEnoyl acyl carrier protein reductase enzyme (ENR) catalyses one of the two reductive steps in fatty acid elongation within the fatty acid synthesis type II cycle that is common to plants and prokaryotes. Since enzymes of this pathway are absent in humans they have become the target for several potent antibacterial compounds including triclosan which inhibits ENR in the picomolar range.
As part of this thesis the gene for a type II ENR was located in the genomes of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Analysis of the derived protein sequences suggested that these enzyme reside in the apicoplast. X-ray crystallographic techniques have been used to solve the structure for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg) ENR in complex with the NAD+ cofactor and triclosan by molecular replacement to 2.2A and 2.6A, respectively. Both enzymes. are tetrameric with the approximate dimensions of 90A x 90A x 50A. Each subunit is formed by a 7 stranded parallel β-sheet flanked by 9α helices, reminiscent of a Rossmann nucleotide binding fold common to several NAD+ binding enzymes. Analysis of the ENR family reveals that a characteristic of apicomplexan ENRs is an insert which varies in size from 42 residues in the P jalciparum enzyme to 6 residues in T.gondii ENR and which flanks the inhibitor/substrate binding site. In PfENR this loop is disordered but in the structure of TgENR the loop can be clearly seen and the structure shows that the loop lies close to the bound inhibitor but makes no direct contacts.
Comparisons of the binding sites of a range of different ENR inhibitor complexes has led to a better understanding of the plasticity of the enzyme in response to inhibitor (and possibly substrate) binding. Moreover analysis of the substrate/inhibitor binding pocket in P jalciparum and T.gondii ENR shows that whilst they are similar to the bacterial enzymes there are distinct differences which could be exploited for the development of novel antiparasitic agents. A major hurdle in the delivery of inhibitors targeted towards the apicoplast organelle is the need to cross several barriers including the parasite membranes and host cell walls. However the addition of a releasable eight arginine linker to the phenolic OH group of triclosan significantly improved the speed of delivery and enabled triclosan to enter both the extracellular and intracellular T.gondii tachyzoites and bradyzoites. The identification of both a novel inhibitor for the apicomplexan family and a possible general delivery mechanism may provide a foundation for the development of ENR inhibitors that will efficiently treat several key parasitic diseases
Asymptotic properties of dynamic stochastic parameter estimates (III)
No full textAbstractIn this paper we establish three theorems concerning the asymptotic distributions of ordinary least-squares estimates of the parameters of a stochastic difference equation. We show that, if there is at least one root of the associated characteristic equation with modulus less than one and if all the roots have moduli different from one, the vector of least-squares estimates converges in distribution to a normally distributed vector. The distribution of the limiting vector is degenerate if there is at least one root with modulus greater than one. The results we obtain represent extensions of results proviously obtained by H. B. Mann and A. Wald, H. Rubin, J. S. White, T. W. Anderson, M. M. Rao, T. J. Muench, and the author
The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy.
No full textDespite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function
Water, Sanitation and the Modern City: Colonial and Post-colonial Experiences in Lagos and Mumbai
No full texthuman development, water, sanitation
-ATPase
No full textThe V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ∼30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies. © 2013 Song et al
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