Inward-directed current generated by the Na+,K+ pump in Na(+)- and K(+)-free medium

In Na(+)- and K(+)-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardiac glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na+,K+ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H+] in the external medium. This current is not observed when Mg2+ instead of Ba2+ is the only divalent cation present in the bath medium, and it does not depend on whether Na+ or K+ is present internally. At 5 to 10 mM Na+ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na+]. It is suggested that in low-Na+ and K(+)-free medium the Na+,K+ pump molecule can either form a conductive pathway that is permeable to Ba2+ or protons or operate in its conventional transport mode accepting Ba2+ as a K+ congener. A reversed pump mode or an electrogenic uncoupled Na(+)-efflux mode is excluded.     

Do additive manufactured parts deserve better?

Additive manufacturing of metallic components is regarded as one of the more exciting developments in engineering. The combined attractions of near net shape, tailored composition, and geometry optimisation have led to much interest in the various processes used and a drive to improve the mechanical properties to match those of wrought parts. In this paper, we reflect on the apparent lack of ambition in optimising the structural integrity of parts made using these new manufacturing processes. The current research focus seems to be either on largely irrelevant static properties, or on quantifying the fatigue response in a way that would be familiar to engineers in the 19^th Century. Given the work on the role of microstructure and fatigue, which dates back to Ewing and Humphrey in 1903 reaching its zenith in the 1980s and 90s with Keith Miller in the vanguard, and recent developments in both imaging technologies and sophisticated numerical modelling, all the elements are in place for a much more rigorous, and ultimately more fruitful, approach to understand the structural integrity of additive manufactured components.     

The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties

The different classes of conventional nuclear localization sequences (NLSs) resemble one another in that NLS-dependent nuclear protein import is energy-dependent and mediated by the cytosolic NLS-binding importin/karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Based on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat using in vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous beta-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no significant effect on the nuclear import kinetics implying that the Tat-NLS was able to confer nuclear accumulation through a pathway distinct from conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-beta-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred binding to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked in the absence of ATP, as well as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to confer nuclear entry and binding to nuclear components. These unique properties indicate that Tat accumulates in the nucleus through a novel import pathway.     

Fatigue crack initiation in AA2024: A coupled micromechanical testing and crystal plasticity study

A new combined experimental and modelling approach has been developed in order to understand the physical mechanisms that lead to crack nucleation in a polycrystalline aluminium alloy AA2024 undergoing cyclic loading. Four‐point bending low‐cycle fatigue tests were performed inside the chamber of a scanning electron microscope on specimens with a through‐thickness central hole, introduced to localize stresses and strains in a small region on the top surface of the sample. Fatigue crack initiation and small crack growth mechanisms were analyzed through high‐resolution scanning electron microscope images, local orientation measurements using electron‐back‐scattered‐diffraction, and local strain measurements using digital image correlation. A crystal plasticity finite element model was developed to simulate the cyclic deformation behaviour of AA2024. Two‐dimensional Voronoi‐based microstructures were generated, and the material parameters for the constitutive equations (including both isotropic and kinematic hardening) were identified using monotonic and fully reversed cyclic tests. A commonly used fatigue crack initiation criterion found in the literature, the maximum accumulated plastic slip, was evaluated in the crystal plasticity finite element model but could not predict the formation of cracks away from the edge of the hole in the deformed specimens. A new criterion combining 2 parameters: The maximum accumulated slip over each individual (critical) slip system and the maximum accumulated slip over all slip systems were formulated to reproduce the experimental locations of crack nucleation in the microstructure.     

Simulation of protective relay performance under short-circuit andtransient swing conditions

Most methods of relay coordination are based on fixed values of fault current for setting protective relays, regardless of the fact that fault currents are time-dependent. The results of alternative transient calculation procedures are presented. These take into consideration the decay of current with time and can therefore be used to identify the sensitivity of the coordination setting and potential cases of difficulty. Comparison of the static and dynamic short-circuit calculation procedures shows that the dynamic procedure, with enhanced machine modeling, produces results that agree well with national standards and minimizes the engineering design effort, with only a small increase in computational overhead. The results presented are used to compare calculated time overcurrent relay settings and tripping times, based on the dynamic short-circuit calculation, with fault clearing times obtained by modeling the relays using a traditional step-by-step dynamic analysis procedure. These two methods of calculation yield results that are in close agreement. It is also shown that inclusion of protection setting and transient modeling facilities in power system CAD packages offers the opportunity to include automatically and reliably the effect of the protection in any subsequent transient studies of the system     

A comparison of plan-based and abstract MDP reward shaping

Reward shaping has been shown to significantly improve an agent's performance in reinforcement learning. As attention is shifting away from tabula-rasa approaches many different reward shaping methods have been developed. In this paper, we compare two different methods for reward shaping; plan-based, in which an agent is provided with a plan and extra rewards are given according to the steps of the plan the agent satisfies, and reward shaping via abstract Markov decision process (MDPs), in which an abstract high-level MDP of the environment is solved and the resulting value function is used to shape the agent. The comparison is conducted in terms of total reward, convergence speed and scaling up to more complex environments. Empirical results demonstrate the need to correctly select and set up reward shaping methods according to the needs of the environment the agents are acting in. This leads to the more interesting question, is there a reward shaping method which is universally better than all other approaches regardless of the environment dynamics?     

Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro. A comparison with the SV40 large T-antigen NLS

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.