Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

A full pedigree based method for the statistical assessment of genetic anticipation

Genetic anticipation is defined as a decrease in age of onset or increase in severity as the disorder is transmitted through subsequent generations. Anticipation has been noted in the literature for over a century. Recently, anticipation in several diseases including Huntington's Disease, Myotonic Dystrophy and Fragile X Syndrome were shown to be caused by expansion of triplet repeats. Anticipation effects have also been observed in numerous mental disorders (e.g. Schizophrenia, Bipolar Disorder), cancers (Li-Fraumeni Syndrome, Leukemia) and other complex diseases. ^ Several statistical methods have been applied to determine whether anticipation is a true phenomenon in a particular disorder, including standard statistical tests and newly developed affected parent/affected child pair methods. These methods have been shown to be inappropriate for assessing anticipation for a variety of reasons, including familial correlation and low power. Therefore, we have developed family-based likelihood modeling approaches to model the underlying transmission of the disease gene and penetrance function and hence detect anticipation. These methods can be applied in extended families, thus improving the power to detect anticipation compared with existing methods based only upon parents and children. The first method we have proposed is based on the regressive logistic hazard model. This approach models anticipation by a generational covariate. The second method allows alleles to mutate as they are transmitted from parents to offspring and is appropriate for modeling the known triplet repeat diseases in which the disease alleles can become more deleterious as they are transmitted across generations. ^ To evaluate the new methods, we performed extensive simulation studies for data simulated under different conditions to evaluate the effectiveness of the algorithms to detect genetic anticipation. Results from analysis by the first method yielded empirical power greater than 87% based on the 5% type I error critical value identified in each simulation depending on the method of data generation and current age criteria. Analysis by the second method was not possible due to the current formulation of the software. The application of this method to Huntington's Disease and Li-Fraumeni Syndrome data sets revealed evidence for a generation effect in both cases. ^

Assessing the landscape value of public works: validation of the method in the lowlands of the middle section of the Tajo river (Spain)

This paper proposes a method of landscape characterisation and assessment of public works associated with fluvial landscapes, which is validated in the middle section of the Tajo River. In this method, a set of criteria is identified that unifies various characteristics of the landscape associated to the infrastructures. A specific weight is then assigned to each criterion in such a way as to produce a semi-quantitative value ranging from a minimum value of 0 to a maximum value of 10. Taken together, these criteria enable us to describe and assess the value of the public works selected for study, in this case helping us to evaluate the sections of the River Tajo analysed in our study area. Accordingly, the value of all the infrastructures associated to a stretch of the river covering several hundred kilometres was determined and after dividing this stretch into sections, they were compared under equivalent conditions to provide a hierarchal ranking.

The transient behaviour of plain concrete at elevated temperatures

This thesis describes an investigation of the effect of elevated temperatures upon the properties of plain concrete containing a siliceous aggregate. A complete stress-strain relationship and creep behaviour are studied. Transient effects (non-steady state) are also examined in order to simulate more realistic conditions. A temperature range of 20-700ºC is used. corresponding to the temperatures generally attained during an actual fire. In order to carry out the requisite tests, a stiff compression testing machine has been designed and built. The overall control of the test rig is provided by a logger/computer system by developing appropriate software, thus enabling the load to be held constant for any period of tlme. Before outlining any details of the development of the testing apparatus which includes an electric furnace and the.associated instrumentation, previous work on properties of both concrete and. steel at elevated temperatures is reviewed. The test programme comprises four series of tests:stress-strain tests (with and without pre-load), transient tests (heating to failure under constant stress) and creep tests (constant stress and constant temperature). Where 3 stress levels are examined: 0.2, 0.4 & 0.6 fc. The experimental results show that the properties of concrete are significantly affected by temperature and the magnitude of the load. The slope of the descending portion branch of the stress-strain curves (strain softening) is found to be temperature dependent. After normalizing the data, the stress-strain curves for different temperatures are represented by a single curve. The creep results are analysed using an approach involving the activation energy which is found to be constant. The analysis shows that the time-dependent deformation is sensibly linear with the applied stress. The total strain concept is shown to hold for the test data within limits.

(Table 1, page 1376) Chemical analyses of manganese nodules and crusts from the Pacific Ocean

A compilation of chemical analyses of Pacific Ocean nodules using an x-ray fluorescence technique. The equipment used was a General Electric XRD-5 with a tungsten tube. Lithium fluoride was used as the diffraction element in assaying for all elements above calcium in the atomic table and EDDT was used in conjunction with a helium path for all elements with an atomic number less than calcium. Flow counters were used in conjunction with a pulse height analyzer to eliminate x-ray lines of different but integral orders in gathering count data. The stability of the equipment was found to be excellent by the author. The equipment was calibrated by the use of standard ores made from pure oxide forms of the elements in the nodules and carefully mixed in proportion to the amounts of these elements generally found in the manganese nodules. Chemically analyzed standards of the nodules themselves were also used. As a final check, a known amount of the element in question was added to selected samples of the nodules and careful counts were taken on these samples before and after the addition of the extra amount of the element. The method involved the determination and subsequent use of absorption and activation factors for the lines of the various elements. All the absorption and activation factors were carefully determined using the standard ores. The chemically analyzed samples of the nodules by these methods yielded an accuracy to at least three significant figures.

The Promise of Access: Hope and Inequality in the Information Economy

In 2013, a series of posters began appearing in Washington, DC’s Metro system. Each declared “The internet: Your future depends on it” next to a photo of a middle-aged black Washingtonian, and an advertisement for the municipal government’s digital training resources. This hopeful discourse is familiar but where exactly does it come from? And how are our public institutions reorganized to approach the problem of poverty as a problem of technology? The Clinton administration’s ‘digital divide’ policy program popularized this hopeful discourse about personal computing powering social mobility, positioned internet startups as the ‘right’ side of the divide, and charged institutions of social reproduction such as schools and libraries with closing the gap and upgrading themselves in the image of internet startups. After introducing the development regime that builds this idea into the urban landscape through what I call the ‘political economy of hope’, and tracing the origin of the digital divide frame, this dissertation draws on three years of comparative ethnographic fieldwork in startups, schools, and libraries to explore how this hope is reproduced in daily life, becoming the common sense that drives our understanding of and interaction with economic inequality and reproduces that inequality in turn. I show that the hope in personal computing to power social mobility becomes a method of securing legitimacy and resources for both white émigré technologists and institutions of social reproduction struggling to understand and manage the persistent poverty of the information economy. I track the movement of this common sense between institutions, showing how the political economy of hope transforms them as part of a larger development project. This dissertation models a new, relational direction for digital divide research that grounds the politics of economic inequality with an empirical focus on technologies of poverty management. It demands a conceptual shift that sees the digital divide not as a bug within the information economy, but a feature of it.

Maximum principle and numerical method for the multi-term time–space Riesz–Caputo fractional differential equations

The maximum principle for the space and time–space fractional partial differential equations is still an open problem. In this paper, we consider a multi-term time–space Riesz–Caputo fractional differential equations over an open bounded domain. A maximum principle for the equation is proved. The uniqueness and continuous dependence of the solution are derived. Using a fractional predictor–corrector method combining the L1 and L2 discrete schemes, we present a numerical method for the specified equation. Two examples are given to illustrate the obtained results.

Titanium in Dentistry: Historical development, state of the art and future perspectives

Titanium is a metallic element known by several attractive characteristics, such as biocompatibility, excellent corrosion resistance and high mechanical resistance. It is widely used in Dentistry, with high success rates, providing a favorable biological response when in contact with live tissues. Therefore, the objective of this study was to describe the different uses of titanium in Dentistry, reviewing its historical development and discoursing about its state of art and future perspective of its utilization. A search in the MEDLINE/PubMed database was performed using the terms 'titanium', 'dentistry' and 'implants'. The title and abstract of articles were read, and after this first screening 20 articles were selected and their full-texts were downloaded. Additional text books and manual search of reference lists within selected articles were included. Correlated literature showed that titanium is the most used metal in Implantology for manufacturing osseointegrated implants and their systems, with a totally consolidated utilization. Moreover, titanium can be also employed in prosthodontics to obtain frameworks. However, problems related to its machining, casting, welding and ceramic application for dental prosthesis are still limiting its use. In Endodontics, titanium has been used in association to nickel for manufacturing rotatory instruments, providing a higher resistance to deformation. However, although the different possibilities of using titanium in modern Dentistry, its use for prostheses frameworks still needs technological improvements in order to surpass its limitations. © 2012 Indian Prosthodontic Society.

The red badge of courage: uma análise descritiva de suas traduções no Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural performance of reinforced concrete beams repairing from spalling

The effectiveness of a repair work for the restoration of spalled reinforced concrete (r.c.) structures depends to a great extent, on their ability to restore the structural integrity of the r.c. element, to restore its serviceability and to protect the reinforcements from further deterioration. This paper presents results of a study concocted to investigate the structural performance of eight spalled r.c. beams repaired using two advanced repair materials in various zones for comparison purposes, namely a free flowing self compacting mortar (FFSCM) and a polymer Modified cementitious mortar (PMCM). The repair technique adopted was that for the repair of spalled concrete in which the bond between the concrete and steel was completely lost due to reinforcement corrosion or the effect of fire or impact. The beams used for the experiment were first cast, then hacked at various zones before they were repaired except for the control beam. The beam specimens were then loaded to failure under four point loadings. The structural response of each beam was evaluated in terms of first crack load, cracking behavior, crack pattern, deflection, variation of strains in the concrete and steel, collapse load and the modes of failure. The results of the test showed that, the repair materials applied on the various zones of the beams were able to restore more than 100% of the beams’ capacity and that FFSCM gave a better overall performance.

Stiffness-damping matching method of an ECAS system based on LQG control

A novel method of matching stiffness and continuous variable damping of an ECAS (electronically controlled air suspension) based on LQG (linear quadratic Gaussian) control was proposed to simultaneously improve the road-friendliness and ride comfort of a two-axle school bus. Taking account of the suspension nonlinearities and target-height-dependent variation in suspension characteristics, a stiffness model of the ECAS mounted on the drive axle of the bus was developed based on thermodynamics and the key parameters were obtained through field tests. By determining the proper range of the target height for the ECAS of the fully-loaded bus based on the design requirements of vehicle body bounce frequency, the control algorithm of the target suspension height (i.e., stiffness) was derived according to driving speed and road roughness. Taking account of the nonlinearities of a continuous variable semi-active damper, the damping force was obtained through the subtraction of the air spring force from the optimum integrated suspension force, which was calculated based on LQG control. Finally, a GA (genetic algorithm)-based matching method between stepped variable damping and stiffness was employed as a benchmark to evaluate the effectiveness of the LQG-based matching method. Simulation results indicate that compared with the GA-based matching method, both dynamic tire force and vehicle body vertical acceleration responses are markedly reduced around the vehicle body bounce frequency employing the LQG-based matching method, with peak values of the dynamic tire force PSD (power spectral density) decreased by 73.6%, 60.8% and 71.9% in the three cases, and corresponding reduction are 71.3%, 59.4% and 68.2% for the vehicle body vertical acceleration. A strong robustness to variation of driving speed and road roughness is also observed for the LQG-based matching method.

The renormalization group: A perturbation method for the graduate curriculum

In this paper the renormalization group (RG) method of Chen, Goldenfeld, and Oono [Phys. Rev. Lett., 73 (1994), pp.1311-1315; Phys. Rev. E, 54 (1996), pp.376-394] is presented in a pedagogical way to increase its visibility in applied mathematics and to argue favorably for its incorporation into the corresponding graduate curriculum.The method is illustrated by some linear and nonlinear singular perturbation problems. Key word. © 2012 Society for Industrial and Applied Mathematics.

Research on Bearing Capacity Calculation Method of Large Diameter Cylinder in Soft Clay

A large diameter cylinder inserted in soils is a new type of engineering structures used in offshore and port engineering. The mechanism of its bearing capacity and the analysis of its stability are important to its design and applications. In this paper, the finite element method is used to analyze the reacting forces of the soft soil foundation on the structure under the wave action. A simplified method is proposed, based on the plastic limit method, for the safety and stability analysis. Our analysis shows that the assumptions made in this paper and the mechanism used are reasonable, and the results obtained are appropriate. The calculation method is very efficient and can be used to evaluate main parameters of the structure in its preliminary designs.

beta-delayed proton decays of Sm-133 and Yb-149

Sm-133 was produced via fusion evaporation in the reaction Ca-40+Ru-96. Its P-delayed proton decay was studied by means of "p-gamma" coincidence in combination with a He-jet tape transport system, including half-lives, proton energy spectra, gamma-transitions following the proton emissions, and the branching ratios to the low-lying states in the grand-daughter nuclei. The possible spins and parities of 133Sm were extracted by fitting the experimental data with a statistical model calculation. The configuration-constrained nuclear potential energy surfaces of Sm-133 were calculated by using the Woods-Saxon Strutinsky method. Comparing the experimental and calculated results, the spins and parities Of Sm-133 were assigned to be 5/2(+) and 1/2(-), which is reconciled with our published simple (EC+beta(+)) decay scheme Of Sm-113 in 2001. In addition, our experimental data on the beta-delayed proton decay of Yb-149 reported in Eur. Phys. J., 2001, A12: 1-4 was also analyzed by using the same method. The spin and parity of Yb-149 was assigned to be 1/2-.