A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr178-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr328-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr178-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr328-) phosphorylation. Interestingly, MST3(Thr328-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr178- /Thr328-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr328-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

The role of prophage-like elements in the diversity of Salmonella enterica serovars

The Salmonella enterica serovar Typhi CT18 (S. Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S. Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S. Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S. Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S. Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S. enterica. (C) 2004 Elsevier Ltd. All rights reserved.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

Motion planning of autonomous vehicles in a non-autonomous vehicle environment without speed lanes

Planning is one of the key problems for autonomous vehicles operating in road scenarios. Present planning algorithms operate with the assumption that traffic is organised in predefined speed lanes, which makes it impossible to allow autonomous vehicles in countries with unorganised traffic. Unorganised traffic is though capable of higher traffic bandwidths when constituting vehicles vary in their speed capabilities and sizes. Diverse vehicles in an unorganised exhibit unique driving behaviours which are analysed in this paper by a simulation study. The aim of the work reported here is to create a planning algorithm for mixed traffic consisting of both autonomous and non-autonomous vehicles without any inter-vehicle communication. The awareness (e.g. vision) of every vehicle is restricted to nearby vehicles only and a straight infinite road is assumed for decision making regarding navigation in the presence of multiple vehicles. Exhibited behaviours include obstacle avoidance, overtaking, giving way for vehicles to overtake from behind, vehicle following, adjusting the lateral lane position and so on. A conflict of plans is a major issue which will almost certainly arise in the absence of inter-vehicle communication. Hence each vehicle needs to continuously track other vehicles and rectify plans whenever a collision seems likely. Further it is observed here that driver aggression plays a vital role in overall traffic dynamics, hence this has also been factored in accordingly. This work is hence a step forward towards achieving autonomous vehicles in unorganised traffic, while similar effort would be required for planning problems such as intersections, mergers, diversions and other modules like localisation.

An exact local conservation theorem for finite-amplitude disturbances to non-parallel shear flows, with remarks on Hamiltonian structure and on Arnol'd's stability theorems

Disturbances of arbitrary amplitude are superposed on a basic flow which is assumed to be steady and either (a) two-dimensional, homogeneous, and incompressible (rotating or non-rotating) or (b) stably stratified and quasi-geostrophic. Flow over shallow topography is allowed in either case. The basic flow, as well as the disturbance, is assumed to be subject neither to external forcing nor to dissipative processes like viscosity. An exact, local ‘wave-activity conservation theorem’ is derived in which the density A and flux F are second-order ‘wave properties’ or ‘disturbance properties’, meaning that they are O(a2) in magnitude as disturbance amplitude a [rightward arrow] 0, and that they are evaluable correct to O(a2) from linear theory, to O(a3) from second-order theory, and so on to higher orders in a. For a disturbance in the form of a single, slowly varying, non-stationary Rossby wavetrain, $\overline{F}/\overline{A}$ reduces approximately to the Rossby-wave group velocity, where (${}^{-}$) is an appropriate averaging operator. F and A have the formal appearance of Eulerian quantities, but generally involve a multivalued function the correct branch of which requires a certain amount of Lagrangian information for its determination. It is shown that, in a certain sense, the construction of conservable, quasi-Eulerian wave properties like A is unique and that the multivaluedness is inescapable in general. The connection with the concepts of pseudoenergy (quasi-energy), pseudomomentum (quasi-momentum), and ‘Eliassen-Palm wave activity’ is noted. The relationship of this and similar conservation theorems to dynamical fundamentals and to Arnol'd's nonlinear stability theorems is discussed in the light of recent advances in Hamiltonian dynamics. These show where such conservation theorems come from and how to construct them in other cases. An elementary proof of the Hamiltonian structure of two-dimensional Eulerian vortex dynamics is put on record, with explicit attention to the boundary conditions. The connection between Arnol'd's second stability theorem and the suppression of shear and self-tuning resonant instabilities by boundary constraints is discussed, and a finite-amplitude counterpart to Rayleigh's inflection-point theorem noted

The effect of non-uniform radiative damping on the zonal-mean dynamics of the extratropical middle atmosphere

The effect of spatial and temporal variations in the radiative damping rate on the response to an imposed forcing or diabatic heating is examined in a zonal-mean model of the middle atmosphere. Attention is restricted to the extratropics, where a linear approach is viable. It is found that regions with weak radiative damping rates are more sensitive in terms of temperature to the remote influence of the diabatic circulation. The delay in the response in such regions can mean that ‘downward’ control is not achieved on seasonal time-scales. A seasonal variation in the radiative damping rate modulates the evolution of the response and leaves a transient-like signature in the annual mean temperature field. Several idealized examples are considered, motivated by topical questions. It is found that wave drag outside the polar vortex can significantly affect the temperatures in its interior, so that high-latitude, high-altitude gravity-wave drag is not the only mechanism for warming the southern hemisphere polar vortex. Diabatic mass transport through the 100 hPa surface is found to lag the seasonal evolution of the wave drag that drives the transport, and thus cannot be considered to be in the downward control regime. On the other hand, the seasonal variation of the radiative damping rate is found to make only a weak contribution to the annual mean temperature increase that has been observed above the ozone hole. Copyright © 2002 Royal Meteorological Society.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria X ananassa Duch.) fruit, a non-climacteric tissue

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

Flow structure and near-field dispersion in arrays of building-like obstacles

Dispersion in the near-field region of localised releases in urban areas is difficult to predict because of the strong influence of individual buildings. Effects include upstream dispersion, trapping of material into building wakes and enhanced concentration fluctuations. As a result, concentration patterns are highly variable in time and mean profiles in the near field are strongly non-Gaussian. These aspects of near-field dispersion are documented by analysing data from direct numerical simulations in arrays of building-like obstacles and are related to the underlying flow structure. The mean flow structure around the buildings is found to exert a strong influence over the dispersion of material in the near field. Diverging streamlines around buildings enhance lateral dispersion. Entrainment of material into building wakes in the very near field gives rise to secondary sources, which then affect the subsequent dispersion pattern. High levels of concentration fluctuations are also found in this very near field; the fluctuation intensity is of order 2 to 5.

Ion flows and heating at a contracting polar-cap boundary: GISMOS observations indicating viscous-like interaction on the flanks of the magnetotail

This paper complements that in this issue by Clauer et al. concerning the international GISMOS campaign of 3–5 June 1987. From a detailed study of the EISCAT data, the polar-cap boundary, as defined by an almost shear east-west convection reversal, is found to contract across the EISCAT field of view between 04 and 07 MLT. An annulus of enhanced ion temperature and non-thermal plasma is observed immediately equatorward of the contracting boundary due to the lag in the response of the neutral-wind pattern to the change in ion flows. The ion flow inside the polar cap and at the boundary is shown to be relatively smooth, compared with that in the auroral oval, at 15-second resolution. The flow at the boundary is directed poleward, with velocities which exceed that of the boundary itself. The effect of velocity shears on the beamswinging technique used to derive the ion flows has been analysed in detail and it is found that spurious flows across a moving boundary can be generated. However, these are much smaller than the observed flows into the polar cap and cannot explain the 7 kV potential difference across the observed segment of the cap boundary between 04:30–06:30 UT. The ion temperature enhancements at the two observing azimuths is used to define the boundary orientation. The results are consistent with recent observations of slow anti-sunward flow of closed field lines on the flanks of the geomagnetic tail, which appears to be generated by some form of “viscous” coupling to the magnetosheath plasma.

Scattered power from non-thermal, F-region plasma observed by EISCAT—evidence for coherent echoes?

Three rapid, poleward bursts of plasma flow, observed by the U.K.-POLAR EISCAT experiment, are studied in detail. In all three cases the large ion velocities (> 1 kms−1) are shown to drive the ion velocity distribution into a non-Maxwellian form, identified by the characteristic shape of the observed spectra and the fact that analysis of the spectra with the assumption of a Maxwellian distribution leads to excessive rises in apparent ion temperature, and an anticorrelation of apparent electron and ion temperatures. For all three periods the total scattered power is shown to rise with apparent ion temperature by up to 6 dB more than is expected for an isotropic Maxwellian plasma of constant density and by an even larger factor than that expected for non-thermal plasma. The anomalous increases in power are only observed at the lower altitudes (< 300 km). At greater altitudes the rise in power is roughly consistent with that simulated numerically for homogeneous, anisotropic, non-Maxwellian plasma of constant density, viewed using the U.K.-POLAR aspect angle. The spectra at times of anomalously high power are found to be asymmetric, showing an enhancement near the downward Doppler-shifted ion-acoustic frequency. Although it is not possible to eliminate completely rapid plasma density fluctuations as a cause of these power increases, such effects cannot explain the observed spectra and the correlation of power and apparent ion temperature without an unlikely set of coincidences. The observations are made along a beam direction which is as much as 16.5° from orthogonality with the geomagnetic field. Nevertheless, some form of coherent-like echo contamination of the incoherent scatter spectrum is the most satisfactory explanation of these data.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

The Acquisition of Aspect in L2 Portuguese & Spanish: Exploring Native/Non-Native Performance Differences.

This study investigates the possibility of native-like ultimate attainment by analyzing L2 knowledge of aspect as seen in the Preterit/Imperfect contrast of highly successful English L2 learners of Portuguese and Spanish. Building on innovative work by Montrul & Slabakova (2003) and Slabakova & Montrul (2003), we test knowledge of semantic entailments associated with the acquisition of [+/- perfective] features checked in higher AspP. Additionally, we investigate the possibility of a specific pattern of associated target-deviant L2 performance. We hypothesize that L2 performance can be affected by explicit positive evidence (pedagogical rules) despite otherwise demonstrable native-like competence. Indeed, the data reveal a pattern of target-deviant performance noted only in three specific contexts, all of which can be linked to traditional instruction: (a) with particular stative verbs not used in the Preterit (b) when preceded by certain adverbial phrases (e.g.,siempre) and (c) so-called semantic shifting verbs (e.g., sabía vs. supe).

Concepts for a geostationary-like polar missions

An evidence-led scientific case for development of a space-based polar remote sensing platform at geostationary-like (GEO-like) altitudes is developed through methods including a data user survey. Whilst a GEO platform provides a nearstatic perspective, multiple platforms are required to provide circumferential coverage. Systems for achieving GEO-like polar observation likewise require multiple platforms however the perspective is non-stationery. A key choice is between designs that provide complete polar view from a single platform at any given instant, and designs where this is obtained by compositing partial views from multiple sensors. Users foresee an increased challenge in extracting geophysical information from composite images and consider the use of non-composited images advantageous. Users also find the placement of apogee over the pole to be preferable to the alternative scenarios. Thus, a clear majority of data users find the “Taranis” orbit concept to be better than a critical inclination orbit, due to the improved perspective offered. The geophysical products that would benefit from a GEO-like polar platform are mainly estimated from radiances in the visible/near infrared and thermal parts of the electromagnetic spectrum, which is consistent with currently proven technologies from GEO. Based on the survey results, needs analysis, and current technology proven from GEO, scientific and observation requirements are developed along with two instrument concepts with eight and four channels, based on Flexible Combined Imager heritage. It is found that an operational system could, mostly likely, be deployed from an Ariane 5 ES to a 16-hour orbit, while a proof-of-concept system could be deployed from a Soyuz launch to the same orbit.

Comparison of terrain following and cut cell grids using a non-hydrostatic model

Terrain following coordinates are widely used in operational models but the cut cell method has been proposed as an alternative that can more accurately represent atmospheric dynamics over steep orography. Because the type of grid is usually chosen during model implementation, it becomes necessary to use different models to compare the accuracy of different grids. In contrast, here a C-grid finite volume model enables a like-for-like comparison of terrain following and cut cell grids. A series of standard two-dimensional tests using idealised terrain are performed: tracer advection in a prescribed horizontal velocity field, a test starting from resting initial conditions, and orographically induced gravity waves described by nonhydrostatic dynamics. In addition, three new tests are formulated: a more challenging resting atmosphere case, and two new advection tests having a velocity field that is everywhere tangential to the terrain following coordinate surfaces. These new tests present a challenge on cut cell grids. The results of the advection tests demonstrate that accuracy depends primarily upon alignment of the flow with the grid rather than grid orthogonality. A resting atmosphere is well-maintained on all grids. In the gravity waves test, results on all grids are in good agreement with existing results from the literature, although terrain following velocity fields lead to errors on cut cell grids. Due to semi-implicit timestepping and an upwind-biased, explicit advection scheme, there are no timestep restrictions associated with small cut cells. We do not find the significant advantages of cut cells or smoothed coordinates that other authors find.