A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein

Recombinant production of HPV oncoprotein E6 is notoriouslydifficult. The unfused sequence is produced in inclusion bodies.By contrast, fusions of E6 to the C-terminus of carrier proteinssuch as maltose-binding protein or gluthatione-S-transferaseare produced soluble. However, it has not yet been possibleto purify E6 protein from such fusion constructs. Here, we showthat this was due to the biophysical heterogeneity of the fusionpreparations. We find that soluble MBP-E6 preparations containtwo subpopulations. A major fraction is aggregated and containsexclusively misfolded E6 moieties (`soluble inclusion bodies').A minor fraction is monodisperse and contains the properly foldedE6 moieties. Using monodispersity as a screening criterion,we optimized the expression conditions, the purification processand the sequence of E6, finally obtaining stable monodisperseMBP-E6 preparations. In contrast to aggregated MBP-E6, thesepreparations yielded fully soluble E6 after proteolytic removalof MBP. Once purified, these E6 proteins are stable, foldedand biologically active. The first biophysical measurementson pure E6 were performed. This work shows that solubility isnot a sufficient criterion to check that the passenger proteinin a fusion construct is properly folded and active. By contrast,monodispersity appears as a better quality criterion. The monodispersity-basedstrategy presented here constitutes a general method to preparefusion proteins with optimized folding and biological activity.     

Optimisation of the Amido Black assay for determination of the protein content of grape juices and wines

While the heat/chill stability of white wines cannot be predicted from the total protein concentration, this information is useful in assessing the effectiveness of fining treatments. We have adapted the Amido Black assay for use in grape juice and wine. The response of bovine serum albumin (BSA) was similar in water, model wine and model juice. The linear range of the assay (<100 mg l^?1) extends beyond protein concentrations typically found in grape juices and wines. The limit of quantification was 11.1 mg l^?1 for BSA in model wine and 7.6 mg l^?1 in model juice, while the limit of detection was found to be less than 3 mg l^?1 in either solution. In contrast to other methods of protein determination, this assay is not affected by common wine phenolic and pectic compounds or by glutathione. The mean response of BSA was similar in model juice and red and white grape juices. The mean response of BSA in red wine was lower than in white wine and model wine (p < 0.001), and there was considerable variation in response among wines. Protein concentrations determined using this method were reproducible (CV < 10%). This optimised assay can be used to monitor protein levels in grape juices and wines. © 2001 Society of Chemical Industry     

Modelling the impacts of CDM incentives for the Thai electricity sector

The CDM Executive Board recently took a positive decision on programmatic CDM, also known as a CDM Programme of Activities. This prompts the author to present a new tool that has been developed recently for the Thai electricity market. The Renewable Energy Development (RED) Model, initially developed in the framework of the DANIDA funded project: Promotion of Renewable Energy in Thailand (PRET), at the Ministry of Energy of Thailand, was designed for the modelling of different incentive schemes and their effects on the Thai power system for the promotion of renewable energy technologies (RETs). Within this article, an extension of the existing RED model, including the CDM as additional incentive measure, is presented (RED-CDM). Along with the project-based approach, also a sectoral and programmatic approach is included as well. Several scenarios developed with the RED-CDM model show the influence of different incentive mechanisms on the Thai power market and their potentials for reaching the policy targets stated in the Energy Strategy of Thailand for Competitiveness. The main results show that reaching the policy targets is possible, while the price can be extremely high if the targets are to be achieved on schedule. Another important result is that a sectoral CDM approach could help financing about 20% of the incentives needed for a shift towards a more sustainable power grid, if the certified emission reductions (CERs) are sold at a price of 15 Euro/ton.     

Reducing the fuel use and greenhouse gas emissions of the US vehicle fleet

The unrelenting increase in the consumption of oil in the US light-duty vehicle fleet (cars and light trucks) presents an extremely challenging energy and environmental problem. A variety of propulsion technologies and fuels have the promise to reduce petroleum use and greenhouse gas emissions from motor vehicles. Even so, achieving a noticeable reduction on both fronts in the near term will require rapid penetration of these technologies into the vehicle fleet, and not all alternatives can meet both objectives simultaneously. Placing a much greater emphasis on reducing fuel consumption rather than improving vehicle performance can greatly reduce the required market penetration rates. Addressing the vehicle performance–size–fuel consumption trade-off should be the priority for policymakers rather than promoting specific vehicle technologies and fuels.     

Applying bottom-up analysis to identify the system boundaries of non-energy use data in international energy statistics

Data on the non-energy use of fossil fuels in energy statistics are subject to major uncertainties. We apply a simple bottom-up methodology to recalculate non-energy use for the entire world and for the 50 countries with the highest consumption of fossil fuels for non-energy purposes. We quantify worldwide non-energy use in the year 2000 to be 24±2 exajoules (EJ), thereby accounting for 6% of the global total primary energy supply (TPES). Our bottom-up estimates are in line with data from international energy statistics for the entire world and for 14 individual countries. Our estimates exceed official non-energy use data for 22 countries, whereas they are lower than official data in the case of 14 countries. Inconsistent system boundaries of non-energy use data in international energy statistics can explain parts of the observed deviations. We regard our bottom-up methodology as reliable albeit being attached with uncertainties. We recommend its use for energy statisticians and greenhouse gas (GHG) inventory makers to generate a shortlist of countries, for which efforts should be made to clarify and improve the quality of non-energy use data in national and international energy statistics.     

On the optimality of solutions of the max-productbelief-propagation algorithm in arbitrary graphs

Graphical models, such as Bayesian networks and Markov random fields (MRFs), represent statistical dependencies of variables by a graph. The max-product “belief propagation” algorithm is a local-message-passing algorithm on this graph that is known to converge to a unique fixed point when the graph is a tree. Furthermore, when the graph is a tree, the assignment based on the fixed point yields the most probable values of the unobserved variables given the observed ones. Good empirical performance has been obtained by running the max-product algorithm (or the equivalent min-sum algorithm) on graphs with loops, for applications including the decoding of “turbo” codes. Except for two simple graphs (cycle codes and single-loop graphs) there has been little theoretical understanding of the max-product algorithm on graphs with loops. Here we prove a result on the fixed points of max-product on a graph with arbitrary topology and with arbitrary probability distributions (discrete- or continuous-valued nodes). We show that the assignment based on a fixed point is a “neighborhood maximum” of the posterior probability: the posterior probability of the max-product assignment is guaranteed to be greater than all other assignments in a particular large region around that assignment. The region includes all assignments that differ from the max-product assignment in any subset of nodes that form no more than a single loop in the graph. In some graphs, this neighborhood is exponentially large. We illustrate the analysis with examples     

Micromilling of microbarbs for medical implants

Successful application of many implantable medical devices often depends on the technique used for attaching the devices to living tissue. Although mechanical attachment techniques that use micro-scale piercing elements are very promising, the fabrication of the micro-scale piercing elements poses considerable challenges. In this paper, the use of the mechanical micromilling process for fabrication of micro-scale piercing elements (microbarbs) from biocompatible materials is investigated. Two micro-scale milling tools with custom geometries are used during the fabrication. To gain an understanding of the process conditions on the performance metrics of micromilling forces, surface roughness, and burr formation, a design of experiments study is performed on polymethyl methacrylate (PMMA). The geometric capability of the process is demonstrated by fabricating PMMA microbarbs with different sizes (widths from 68 to ), edge sharpnesses (60 and 90 included angles), and heights (from 84 to ). The capability to micromill a variety of biocompatible materials is demonstrated by creating microbarbs from non-biodegradable 304 stainless steel and PMMA, from bioresorbable polylactic acid (PLA), and from a fibrin based plastic. It is concluded that the micromilling process can be used for effectively fabricating arrays of microbarbs.     

Boron dilution transients during natural circulation flow in PWR—Experiments and CFD simulations

Coolant mixing in the cold leg, downcomer and the lower plenum of pressurized water reactors is an important phenomenon mitigating the reactivity insertion into the core. Therefore, mixing of the de-borated slugs with the ambient coolant in the reactor pressure vessel was investigated at the four loops 1:5 scaled Rossendorf coolant mixing model (ROCOM) mixing test facility. In particular thermal hydraulics analyses have shown, that weakly borated condensate can accumulate in the pump loop seal of those loops, which do not receive a safety injection. After refilling of the primary circuit, natural circulation in the stagnant loops can re-establish simultaneously and the de-borated slugs are shifted towards the reactor pressure vessel (RPV).In the ROCOM experiments, the length of the flow ramp and the initial density difference between the slugs and the ambient coolant was varied. From the test matrix experiments with 0 resp. 2% density difference between the de-borated slugs and the ambient coolant were used to validate the CFD software ANSYS CFX. To model the effects of turbulence on the mean flow a higher order Reynolds stress turbulence model was employed and a mesh consisting of 6.4 million hybrid elements was utilized. Only the experiments and CFD calculations with modeled density differences show stratification in the downcomer. Depending on the degree of density differences the less dense slugs flow around the core barrel at the top of the downcomer. At the opposite side, the lower borated coolant is entrained by the colder safety injection water and transported to the core. The validation proves that ANSYS CFX is able to simulate appropriately the flow field and mixing effects of coolant with different densities.     

The impact of LOC structures on 670-nm (Al)GaInP high-power lasers

We investigate the potential of large optical cavity (LOC)-laser structures for AlGaInP high-power lasers. For that we study large series of broad area lasers with varying waveguide widths to obtain statistically relevant data. We study in detail I/sub th/, /spl alpha//sub i/, /spl eta//sub i/, and P/sub max/, and analyze above-threshold behavior including temperature stability and leakage current. We got as expected for LOC structures minimal /spl alpha//sub i//spl les/1 cm/sup -1/ resulting in /spl eta//sup d/=1.1 W/A for 64/spl times/2000 /spl mu/m/sup 2/ uncoated devices. We obtain total output powers /spl ges/3.2 W (qCW) and /spl ges/1.5 W (CW) at 20/spl deg/C.