Structure of microtubule-trapped Human Kinesin-5 and its mechanism of inhibition revealed using Cryoelectron Microscopy

Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins

Wernicke's Encephalopathy: 'plus ca change, plus c'est la meme chose'

Aims: To develop clinical guidelines to identify individuals who misuse alcohol and are at risk of developing Wernicke's Encephalopathy (WE). Method: Non-systematic literature review of studies which includes a careful clinical record of the development of signs and symptoms of thiamine deficiency and in which the diagnosis of WE has been confirmed at autopsy. Results: The review of the clinical findings in cases of WE, diagnosed at autopsy, shows a consistent pattern of signs and symptoms. The pattern appears to be similar regardless of whether the thiamine deficiency is related to nutritional problems alone or associated with alcohol misuse. Conclusions: The assessment of the degree of thiamine deficiency and the diagnosis of WE remain a clinical evaluation, and guidelines are suggested to help the clinician. Since neurotoxicity due to the metabolism of excessive alcohol in patients with chronic and severe alcohol dependence may be an important factor in determining long-term outcome of treatment, this must form part of the overall evaluation

Comparing initial-data sets for binary black holes

We compare the results of constructing binary black hole initial data with three different decompositions of the constraint equations of general relativity. For each decomposition we compute the initial data using a superposition of two Kerr-Schild black holes to fix the freely specifiable data. We find that these initial-data sets differ significantly, with the ADM energy varying by as much as 5% of the total mass. We find that all initial-data sets currently used for evolutions might contain unphysical gravitational radiation of the order of several percent of the total mass. This is comparable to the amount of gravitational-wave energy observed during the evolved collision. More astrophysically realistic initial data will require more careful choices of the freely specifiable data and boundary conditions for both the metric and extrinsic curvature. However, we find that the choice of extrinsic curvature affects the resulting data sets more strongly than the choice of conformal metric.Comment: 18 pages, 12 figures, accepted for publication in Phys. Rev.

Corotating and irrotational binary black holes in quasi-circular orbits

A complete formalism for constructing initial data representing black-hole binaries in quasi-equilibrium is developed. Radiation reaction prohibits, in general, true equilibrium binary configurations. However, when the timescale for orbital decay is much longer than the orbital period, a binary can be considered to be in quasi-equilibrium. If each black hole is assumed to be in quasi-equilibrium, then a complete set of boundary conditions for all initial data variables can be developed. These boundary conditions are applied on the apparent horizon of each black hole, and in fact force a specified surface to be an apparent horizon. A global assumption of quasi-equilibrium is also used to fix some of the freely specifiable pieces of the initial data and to uniquely fix the asymptotic boundary conditions. This formalism should allow for the construction of completely general quasi-equilibrium black hole binary initial data.Comment: 13 pages, no figures, revtex4; Content changed slightly to reflect fact that regularized shift solutions do satisfy the isometry boundary condition

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Background and Purpose Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. Experimental Approach We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. Key Results Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). Conclusions and Implications The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses

Primordialists and Constructionists: a typology of theories of religion

This article adopts categories from nationalism theory to classify theories of religion. Primordialist explanations are grounded in evolutionary psychology and emphasize the innate human demand for religion. Primordialists predict that religion does not decline in the modern era but will endure in perpetuity. Constructionist theories argue that religious demand is a human construct. Modernity initially energizes religion, but subsequently undermines it. Unpacking these ideal types is necessary in order to describe actual theorists of religion. Three distinctions within primordialism and constructionism are relevant. Namely those distinguishing: a) materialist from symbolist forms of constructionism; b) theories of origins from those pertaining to the reproduction of religion; and c) within reproduction, between theories of religious persistence and secularization. This typology helps to make sense of theories of religion by classifying them on the basis of their causal mechanisms, chronology and effects. In so doing, it opens up new sightlines for theory and research

Circular orbits of corotating binary black holes: comparison between analytical and numerical results

We compare recent numerical results, obtained within a ``helical Killing vector'' (HKV) approach, on circular orbits of corotating binary black holes to the analytical predictions made by the effective one body (EOB) method (which has been recently extended to the case of spinning bodies). On the scale of the differences between the results obtained by different numerical methods, we find good agreement between numerical data and analytical predictions for several invariant functions describing the dynamical properties of circular orbits. This agreement is robust against the post-Newtonian accuracy used for the analytical estimates, as well as under choices of resummation method for the EOB ``effective potential'', and gets better as one uses a higher post-Newtonian accuracy. These findings open the way to a significant ``merging'' of analytical and numerical methods, i.e. to matching an EOB-based analytical description of the (early and late) inspiral, up to the beginning of the plunge, to a numerical description of the plunge and merger. We illustrate also the ``flexibility'' of the EOB approach, i.e. the possibility of determining some ``best fit'' values for the analytical parameters by comparison with numerical data.Comment: Minor revisions, accepted for publication in Phys. Rev. D, 19 pages, 6 figure

Consistent histories, the quantum Zeno effect, and time of arrival

We present a decomposition of the general quantum mechanical evolution operator, that corresponds to the path decomposition expansion, and interpret its constituents in terms of the quantum Zeno effect (QZE). This decomposition is applied to a finite dimensional example and to the case of a free particle in the real line, where the possibility of boundary conditions more general than those hitherto considered in the literature is shown. We reinterpret the assignment of consistent probabilities to different regions of spacetime in terms of the QZE. The comparison of the approach of consistent histories to the problem of time of arrival with the solution provided by the probability distribution of Kijowski shows the strength of the latter point of view

The Time-Energy Uncertainty Relation

The time energy uncertainty relation has been a controversial issue since the advent of quantum theory, with respect to appropriate formalisation, validity and possible meanings. A comprehensive account of the development of this subject up to the 1980s is provided by a combination of the reviews of Jammer (1974), Bauer and Mello (1978), and Busch (1990). More recent reviews are concerned with different specific aspects of the subject. The purpose of this chapter is to show that different types of time energy uncertainty relation can indeed be deduced in specific contexts, but that there is no unique universal relation that could stand on equal footing with the position-momentum uncertainty relation. To this end, we will survey the various formulations of a time energy uncertainty relation, with a brief assessment of their validity, and along the way we will indicate some new developments that emerged since the 1990s.Comment: 33 pages, Latex. This expanded version (prepared for the 2nd edition of "Time in quantum mechanics") contains minor corrections, new examples and pointers to some additional relevant literatur