Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast

Background and Purpose The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. Experimental Approach We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. Key Results The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. Conclusions and Implications These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Phosphorylation and membrane dissociation of the ARF exchange factor GBF1 in mitosis

Secretory protein trafficking is arrested and the Golgi apparatus fragmented when mammalian cells enter mitosis. These changes are thought to facilitate cell cycle progression and Golgi inheritance, and are brought about through the actions of mitotically active protein kinases. To better understand how the Golgi apparatus undergoes mitotic fragmentation we have sought to identify novel Golgi targets for mitotic kinases. We report here the identification of the ARF exchange factor GBF1 as a Golgi phosphoprotein. GBF1 is phosphorylated by CDK1-cyclin B in mitosis, which results in its dissociation from Golgi membranes. Consistent with a reduced level of GBF1 activity at the Golgi membrane there is a reduction in levels of membrane-associated GTP-bound ARF in mitotic cells. Despite the reduced levels of membrane bound GBF1 and ARF, COPI binding to the Golgi membrane appears unaffected in mitotic cells. Surprisingly, this pool of COPI is dependent upon GBF1 for its recruitment to the membrane, suggesting a low level of GBF1 activity persists in mitosis. We propose that the phosphorylation and membrane dissociation of GBF1 and the consequent reduction in ARF-GTP levels in mitosis are important for changes in Golgi dynamics and possibly other mitotic events mediated through effectors other than the COPI vesicle coat.

Evidence for dissociation between the perceptual and visuomotor systems in humans

When a visual stimulus is continuously moved behind a small stationary window, the window appears displaced in the direction of motion of the stimulus. In this study we showed that the magnitude of this illusion is dependent on (i) whether a perceptual or visuomotor task is used for judging the location of the window, (ii) the directional signature of the stimulus, and (iii) whether or not there is a significant delay between the end of the visual presentation and the initiation of the localization measure. Our stimulus was a drifting sinusoidal grating windowed in space by a stationary, two-dimensional, Gaussian envelope (σ=1 cycle of sinusoid). Localization measures were made following either a short (200 ms) or long (4.2 s) post-stimulus delay. The visuomotor localization error was up to three times greater than the perceptual error for a short delay. However, the visuomotor and perceptual localization measures were similar for a long delay. Our results provide evidence in support of the hypothesis that separate cortical pathways exist for visual perception and visually guided action and that delayed actions rely on stored perceptual information.

A developmental dissociation of view-dependent and view-invariant object recognition in adolescence

Spatial generalization skills in school children aged 8-16 were studied with regard to unfamiliar objects that had been previously learned in a cross-modal priming and learning paradigm. We observed a developmental dissociation with younger children recognizing objects only from previously learnt perspectives whereas older children generalized acquired object knowledge to new viewpoints as well. Haptic and - to a lesser extent - visual priming improved spatial generalization in all but the youngest children. The data supports the idea of dissociable, view-dependent and view-invariant object representations with different developmental trajectories that are subject to modulatory effects of priming. Late-developing areas in the parietal or the prefrontal cortex may account for the retarded onset of view-invariant object recognition. © 2006 Elsevier B.V. All rights reserved.

Constant shear along a boundary of a flume

'I'he accurate rreasurement of bed shear stress has been extremely difficult due to its changing values until white propunded a theory which would give constant shear along the bed of a flume. In this investigation a flume has been designed according to White's theory and by two separate methods proven to give constant shearing force along the bed. The first method applied the Hydrogen Bubble Technique to obtain accurate values of velocity thus allowing the velocity profile to be plotted and the momentum at the various test sections to be calculated. The use of a 16 mm Beaulieu movie camera allowed the exact velocity profiles created by the hydrogen bubbles to be recorded whilst an analysing projector gave the means of calculating the exact velocities at the various test sections. Simultaneously Preston's technique of measuring skin friction using Pitot tubes was applied. Twc banks of open ended water manometer were used for recording the static and velocity head pressure drop along the flume. This tvpe of manometer eliminated air locks in the tubes and was found to be sufficiently accurate. Readings of pressure and velocity were taken for various types and diameters of bed material both natural sands and glass spheres and the results tabulated. Graphs of particle Reynolds Number against bed shear stress were plotted and gave a linear relationship which dropped off at high values of Reynolds number. It was found that bed movement occurred instantaneously along the bed of the flume once critical velocity had been reached. On completion of this test a roof curve inappropriate to the bed material was used and then the test repeated. The bed shearing stress was now no longer constant and yet bed movement started instantaneously along the bed of the flume, showing that there are more parameters than critical shear stress to bed movement. It is concluded from the two separate methods applied that the bed shear stress is constant along the bed of the flume.

Generalized methods and solvers for noise removal from piecewise constant signals. I. Background theory

Removing noise from piecewise constant (PWC) signals is a challenging signal processing problem arising in many practical contexts. For example, in exploration geosciences, noisy drill hole records need to be separated into stratigraphic zones, and in biophysics, jumps between molecular dwell states have to be extracted from noisy fluorescence microscopy signals. Many PWC denoising methods exist, including total variation regularization, mean shift clustering, stepwise jump placement, running medians, convex clustering shrinkage and bilateral filtering; conventional linear signal processing methods are fundamentally unsuited. This paper (part I, the first of two) shows that most of these methods are associated with a special case of a generalized functional, minimized to achieve PWC denoising. The minimizer can be obtained by diverse solver algorithms, including stepwise jump placement, convex programming, finite differences, iterated running medians, least angle regression, regularization path following and coordinate descent. In the second paper, part II, we introduce novel PWC denoising methods, and comparisons between these methods performed on synthetic and real signals, showing that the new understanding of the problem gained in part I leads to new methods that have a useful role to play.

Generalized methods and solvers for noise removal from piecewise constant signals. II. New methods

Removing noise from signals which are piecewise constant (PWC) is a challenging signal processing problem that arises in many practical scientific and engineering contexts. In the first paper (part I) of this series of two, we presented background theory building on results from the image processing community to show that the majority of these algorithms, and more proposed in the wider literature, are each associated with a special case of a generalized functional, that, when minimized, solves the PWC denoising problem. It shows how the minimizer can be obtained by a range of computational solver algorithms. In this second paper (part II), using this understanding developed in part I, we introduce several novel PWC denoising methods, which, for example, combine the global behaviour of mean shift clustering with the local smoothing of total variation diffusion, and show example solver algorithms for these new methods. Comparisons between these methods are performed on synthetic and real signals, revealing that our new methods have a useful role to play. Finally, overlaps between the generalized methods of these two papers and others such as wavelet shrinkage, hidden Markov models, and piecewise smooth filtering are touched on.

A priori prediction of aggregation efficiency and rate constant for fluidized bed melt granulation

This paper presents a predictive aggregation rate model for spray fluidized bed melt granulation. The aggregation rate constant was derived from probability analysis of particle–droplet contact combined with time scale analysis of droplet solidification and granule–granule collision rates. The latter was obtained using the principles of kinetic theory of granular flow (KTGF). The predicted aggregation rate constants were validated by comparison with reported experimental data for a range of binder spray rate, binder droplet size and operating granulator temperature. The developed model is particularly useful for predicting particle size distributions and growth using population balance equations (PBEs).

Build-up of auditory stream segregation induced by tone sequences of constant or alternating frequency and the resetting effects of single deviants

A sequence of constant-frequency tones can promote streaming in a subsequent sequence of alternating-frequency tones, but why this effect occurs is not fully understood and its time course has not been investigated. Experiment 1 used a 2.0-s-long constant-frequency inducer (10 repetitions of a low-frequency pure tone) to promote segregation in a subsequent, 1.2-s test sequence of alternating low- and high-frequency tones. Replacing the final inducer tone with silence substantially reduced reported test-sequence segregation. This reduction did not occur when either the 4th or 7th inducer was replaced with silence. This suggests that a change at the induction/test-sequence boundary actively resets build-up, rather than less segregation occurring simply because fewer inducer tones were presented. Furthermore, Experiment 2 found that a constant-frequency inducer produced its maximum segregation-promoting effect after only three tones—this contrasts with the more gradual build-up typically observed for alternating-frequency sequences. Experiment 3 required listeners to judge continuously the grouping of 20-s test sequences. Constant-frequency inducers were considerably more effective at promoting segregation than alternating ones; this difference persisted for ~10 s. In addition, resetting arising from a single deviant (longer tone) was associated only with constant-frequency inducers. Overall, the results suggest that constant-frequency inducers promote segregation by capturing one subset of test-sequence tones into an ongoing, preestablished stream, and that a deviant tone may reduce segregation by disrupting this capture. These findings offer new insight into the dynamics of stream segregation, and have implications for the neural basis of streaming and the role of attention in stream formation. (PsycINFO Database Record (c) 2013 APA, all rights reserved)

Fractionating the unitary notion of dissociation:disembodied but not embodied dissociative experiences are associated with exocentric perspective-taking

It has been argued that hallucinations which appear to involve shifts in egocentric perspective (e.g., the out-of-body experience, OBE) reflect specific biases in exocentric perspective-taking processes. Via a newly devised perspective-taking task, we examined whether such biases in perspective-taking were present in relation to specific dissociative anomalous body experiences (ABE) - namely the OBE. Participants also completed the Cambridge Depersonalization Scale (CDS; Sierra and Berrios, 2000) which provided measures of additional embodied ABE (unreality of self) and measures of derealization (unreality of surroundings). There were no reliable differences in the level of ABE, emotional numbing, and anomalies in sensory recall reported between the OBE and control group as measured by the corresponding CDS subscales. In contrast, the OBE group did provide significantly elevated measures of derealization ("alienation from surroundings" CDS subscale) relative to the control group. At the same time we also found that the OBE group was significantly more efficient at completing all aspects of the perspective-taking task relative to controls. Collectively, the current findings support fractionating the typically unitary notion of dissociation by proposing a distinction between embodied dissociative experiences and disembodied dissociative experiences - with only the latter being associated with exocentric perspective-taking mechanisms. Our findings - obtained with an ecologically valid task and a homogeneous OBE group - also call for a re-evaluation of the relationship between OBEs and perspective-taking in terms of facilitated disembodied experiences. © 2013 Braithwaite, James, Dewe, Takahashi, Medford and Kessler.

Bifurcation study for a vertical channel with constant flux and large aspect ratio

Using suitable coupled Navier-Stokes Equations for an incompressible Newtonian fluid we investigate the linear and non-linear steady state solutions for both a homogeneously and a laterally heated fluid with finite Prandtl Number (Pr=7) in the vertical orientation of the channel. Both models are studied within the Large Aspect Ratio narrow-gap and under constant flux conditions with the channel closed. We use direct numerics to identify the linear stability criterion in parametric terms as a function of Grashof Number (Gr) and streamwise infinitesimal perturbation wavenumber (making use of the generalised Squire’s Theorem). We find higher harmonic solutions at lower wavenumbers with a resonance of 1:3exist, for both of the heating models considered. We proceed to identify 2D secondary steady state solutions, which bifurcate from the laminar state. Our studies show that 2D solutions are found not to exist in certain regions of the pure manifold, where we find that 1:3 resonant mode 2D solutions exist, for low wavenumber perturbations. For the homogeneously heated fluid, we notice a jump phenomenon existing between the pure and resonant mode secondary solutions for very specific wavenumbers .We attempt to verify whether mixed mode solutions are present for this model by considering the laterally heated model with the same geometry. We find mixed mode solutions for the laterally heated model showing that a bridge exists between the pure and 1:3 resonant mode 2D solutions, of which some are stationary and some travelling. Further, we show that for the homogeneously heated fluid that the 2D solutions bifurcate in hopf bifurcations and there exists a manifold where the 2D solutions are stable to Eckhaus criterion, within this manifold we proceed to identify 3D tertiary solutions and find that the stability for said 3D bifurcations is not phase locked to the 2D state. For the homogeneously heated model we identify a closed loop within the neutral stability curve for higher perturbation wavenumubers and analyse the nature of the multiple 2D bifurcations around this loop for identical wavenumber and find that a temperature inversion occurs within this loop. We conclude that for a homogeneously heated fluid it is possible to have abrup ttransitions between the pure and resonant 2D solutions, and that for the laterally heated model there exist a transient bifurcation via mixed mode solutions.

Build-up of auditory stream segregation induced by tone sequences of constant or alternating frequency and the resetting effects of single deviants

A sequence of constant-frequency tones can promote streaming in a subsequent sequence of alternating-frequency tones, but why this effect occurs is not fully understood and its time course has not been investigated. Experiment 1 used a 2.0-s-long constant-frequency inducer (10 repetitions of a low-frequency pure tone) to promote segregation in a subsequent, 1.2-s test sequence of alternating low- and high-frequency tones. Replacing the final inducer tone with silence substantially reduced reported test-sequence segregation. This reduction did not occur when either the 4th or 7th inducer was replaced with silence. This suggests that a change at the induction/test-sequence boundary actively resets build-up, rather than less segregation occurring simply because fewer inducer tones were presented. Furthermore, Experiment 2 found that a constant-frequency inducer produced its maximum segregation-promoting effect after only three tones—this contrasts with the more gradual build-up typically observed for alternating-frequency sequences. Experiment 3 required listeners to judge continuously the grouping of 20-s test sequences. Constant-frequency inducers were considerably more effective at promoting segregation than alternating ones; this difference persisted for ~10 s. In addition, resetting arising from a single deviant (longer tone) was associated only with constant-frequency inducers. Overall, the results suggest that constant-frequency inducers promote segregation by capturing one subset of test-sequence tones into an ongoing, preestablished stream, and that a deviant tone may reduce segregation by disrupting this capture. These findings offer new insight into the dynamics of stream segregation, and have implications for the neural basis of streaming and the role of attention in stream formation. (PsycINFO Database Record (c) 2013 APA, all rights reserved)

Negative input-resistance compensator for a constant power load

A negative input-resistance compensator is designed to stabilize a power electronic brushless dc motor drive with constant power-load characteristics. The strategy is to feed a portion of the changes in the dc-link voltage into the current control loop to modify the system input impedance in the midfrequency range and thereby to damp the input filter. The design process of the compensator and the selection of parameters are described. The impact of the compensator is examined on the motor-controller performance, and finally, the effectiveness of the controller is verified by simulation and experimental testing.

Effects of short-term detraining on postprandial metabolism, endothelial function, and inflammation in endurance-trained men:dissociation between changes in triglyceride metabolism and endothelial function

Endurance-trained athletes experience a low level of postprandial lipaemia, but this rapidly increases with detraining. We sought to determine whether detraining-induced changes to postprandial metabolism influenced endothelial function and inflammation. Eight endurance-trained men each undertook two oral fat tolerance tests [blood taken fasted and for 6 h following a high-fat test meal (80 g fat, 80 g carbohydrate)]: one during a period of their normal training (trained) and one after 1 wk of no exercise (detrained). Endothelial function in the cutaneous microcirculation was assessed using laser Doppler imaging with iontophoresis in the fasted state and 4 h postprandially during each test. Fasting plasma triglyceride (TG) concentrations increased by 35% with detraining (P = 0.002), as did postprandial plasma (by 53%, P = 0.002), chylomicron (by 68%, P = 0.02) and very low-density lipoprotein (by 51%, P = 0.005) TG concentrations. Endothelial function decreased postprandially in both the trained (by 17%, P = 0.03) and detrained (by 22%, P = 0.03) conditions but did not differ significantly between the trained and detrained conditions in either the fasted or the postprandial states. These results suggest that, although fat ingestion induces endothelial dysfunction, interventions that alter postprandial TG metabolism will not necessarily concomitantly influence endothelial function.

Near-constant time delay response of fibre grating Sagnac loop filters

A theoretical and experimental investigation of the time delay characteristics of fiber Bragg grating-based Sagnac loops (FBGSLs) is presented. Analytic expressions for the phase and time delay of the FBGSL have been derived and excellent agreement is found between their predictions and experimental results for configurations incorporating uniform-period and chirped-period gratings. For symmetrical grating structures, it is found that the FBGSL time delay response is similar to that of the incorporated grating; with asymmetrical gratings, the FBGSL response is quite different. It is shown that wavelength-division-multiplexing filters exhibiting near-zero dispersion characteristics can be implemented using FBGSLs.