A flow reactor process for the synthesis of peptides utilizing immobilized reagents, scavengers and catch and release protocols

A general flow process for the multi-step assembly of peptides has been developed and this procedure has been used to successfully construct a series of Boc, Cbz and Fmoc N-protected dipeptides in excellent yields and purities, including an extension of the method to enable the preparation of a tripeptide derivative.

Exogenous alpha-Synuclein decreases raft partitioning of Cav2.2 channels inducing dopamine release

alpha-Synuclein is thought to regulate neurotransmitter release through multiple interactions with presynaptic proteins, cytoskeletal elements, ion channels, and synaptic vesicles membrane. alpha-Synuclein is abundant in the presynaptic compartment, and its release from neurons and glia has been described as responsible for spreading of alpha-synuclein-derived pathology. alpha-Synuclein-dependent dysregulation of neurotransmitter release might occur via its action on surface-exposed calcium channels. Here, we provide electrophysiological and biochemical evidence to show that alpha-synuclein, applied to rat neurons in culture or striatal slices, selectively activates Cav2.2 channels, and said activation correlates with increased neurotransmitter release. Furthermore, in vivo perfusion of alpha-synuclein into the striatum also leads to acute dopamine release. We further demonstrate that alpha-synuclein reduces the amount of plasma membrane cholesterol and alters the partitioning of Cav2.2 channels, which move from raft to cholesterol-poor areas of the plasma membrane. We provide evidence for a novel mechanism through which alpha-synuclein acts from the extracellular milieu to modulate neurotransmitter release and propose a unifying hypothesis for the mechanism of alpha-synuclein action on multiple targets: the reorganization of plasma membrane microdomains.

Synoptic versus orographic control on stationary convective banding

Quasi-stationary convective bands can cause large localised rainfall accumulations and are often anchored by topographic features. Here, the predictability of and mechanisms causing one such band are determined using ensembles of the Met Office Unified Model at convection-permitting resolution (1.5 km grid length). The band was stationary over the UK for 3 h and produced rainfall accumulations of up to 34 mm. The amount and location of the predicted rainfall was highly variable despite only small differences between the large-scale conditions of the ensemble members. Only three of 21 members of the control ensemble produced a stationary rain band; these three had the weakest upstream winds and hence lowest Froude number. Band formation was due to the superposition of two processes: lee-side convergence resulting from flow around an upstream obstacle and thermally forced convergence resulting from elevated heating over the upstream terrain. Both mechanisms were enhanced when the Froude number was lower. By increasing the terrain height (thus reducing the Froude number), the band became more predictable. An ensemble approach is required to successfully predict the possible occurrence of such quasi-stationary convective events because the rainfall variability is largely modulated by small variations of the large-scale flow. However, high-resolution models are required to accurately resolve the small-scale interactions of the flow with the topography upon which the band formation depends. Thus, although topography provides some predictability, the quasi-stationary convective bands anchored by it are likely to remain a forecasting challenge for many years to come.

Simulated climate change impact on summer dissolved organic carbon release from peat and surface vegetation: implications for drinking water treatment

Uncertainty regarding changes in dissolved organic carbon (DOC) quantity and quality has created interest in managing peatlands for their ecosystem services such as drinking water provision. The evidence base for such interventions is, however, sometimes contradictory. We performed a laboratory climate manipulation using a factorial design on two dominant peatland vegetation types (Calluna vulgaris and Sphagnum Spp.) and a peat soil collected from a drinking water catchment in Exmoor National Park, UK. Temperature and rainfall were set to represent baseline and future conditions under the UKCP09 2080s high emissions scenario for July and August. DOC leachate then underwent standard water treatment of coagulation/flocculation before chlorination. C. vulgaris leached more DOC than Sphagnum Spp. (7.17 versus 3.00 mg g−1) with higher specific ultraviolet (SUVA) values and a greater sensitivity to climate, leaching more DOC under simulated future conditions. The peat soil leached less DOC (0.37 mg g−1) than the vegetation and was less sensitive to climate. Differences in coagulation removal efficiency between the DOC sources appears to be driven by relative solubilisation of protein-like DOC, observed through the fluorescence peak C/T. Post-coagulation only differences between vegetation types were detected for the regulated disinfection by-products (DBPs), suggesting climate change influence at this scale can be removed via coagulation. Our results suggest current biodiversity restoration programmes to encourage Sphagnum Spp. will result in lower DOC concentrations and SUVA values, particularly with warmer and drier summers.

Impact of game bird release on the Adonis blue butterfly Polyommatus bellargus (Lepidoptera Lycaenidae) on chalk grassland

Polyommatus bellargus is a priority species of butterfly in the UK as a result of its scarcity and the rate of population decline over the last few years. In the UK, the species is associated with chalk grassland on hot, south-facing slopes suitable for the growth of the food plant Hippocrepis comosa. Shooting game birds is a popular pastime in the UK. Over 40 million game birds, principally Phasianus colchicus and Alectoris rufa, are bred and released into the countryside each year for shooting interests. There is a concern that the release of such a large number of non-native birds has an adverse effect on native wildlife. A study was carried out over a period of 3 years out to examine whether there was any evidence that A. rufa released into chalk grassland habitat negatively affects populations of P. bellargus. A comparison was made between sites where large numbers of A. rufa were released versus sites where no, or few, birds were released. The study involved the construction of exclosures in these sites to allow an examination of the number of butterflies emerging from H. comosa when the birds were excluded versus when the birds had free range across the area. Where birds were present the on-site vegetation was shorter than where they were absent indicating that the birds were definitely influencing habitat structure. However, the evidence that A. rufa was negatively influencing the number of adult butterflies emerging was not strong, although there was a largely non-significant tendency for higher butterfly emergence when the birds were excluded or absent.

Meteorological forcing data for urban outdoor thermal comfort models from a coupled convective boundary layer and surface energy balance scheme

Site-specific meteorological forcing appropriate for applications such as urban outdoor thermal comfort simulations can be obtained using a newly coupled scheme that combines a simple slab convective boundary layer (CBL) model and urban land surface model (ULSM) (here two ULSMs are considered). The former simulates daytime CBL height, air temperature and humidity, and the latter estimates urban surface energy and water balance fluxes accounting for changes in land surface cover. The coupled models are tested at a suburban site and two rural sites, one irrigated and one unirrigated grass, in Sacramento, U.S.A. All the variables modelled compare well to measurements (e.g. coefficient of determination = 0.97 and root mean square error = 1.5 °C for air temperature). The current version is applicable to daytime conditions and needs initial state conditions for the CBL model in the appropriate range to obtain the required performance. The coupled model allows routine observations from distant sites (e.g. rural, airport) to be used to predict air temperature and relative humidity in an urban area of interest. This simple model, which can be rapidly applied, could provide urban data for applications such as air quality forecasting and building energy modelling, in addition to outdoor thermal comfort.

Relationship of dayside auroral precipitations to the open-closed separatrix and the pattern of convective flow

The implications are discussed of acceleration of magnetospheric ions by reflection off two magnetopause Alfvén waves, launched by the reconnection site into the inflow regions on both sides of the boundary. The effects of these waves on the ion populations, predicted using the model described by Lockwood et al. [1996], offer a physical interpretation of all the various widely used classifications of precipitation into the dayside ionosphere, namely, central plasma sheet, dayside boundary plasma sheet (BPS), void, low-latitude boundary layer (LLBL), cusp, mantle, and polar cap. The location of the open-closed boundary and the form of the convection flow pattern are discussed in relation to the regions in which these various precipitations are typically found. Specifically, the model predicts that both the LLBL and the dayside BPS precipitations are on newly opened field lines and places the convection reversal within the LLBL, as is often observed. It is shown that this offers solutions to a number of paradoxes and problems that arise if the LLBL and BPS precipitations are thought of as being on closed field lines. This model is also used to make quantitive predictions of the longitudinal extent and latitudinal width of the cusp, as a function of solar wind density.

Dependence of convective flows and particle precipitation in the high-latitude dayside ionosphere on theXandYcomponents of the interplanetary magnetic field

The asymmetries in the convective flows, current systems, and particle precipitation in the high-latitude dayside ionosphere which are related to the equatorial plane components of the interplanetary magnetic field (IMF) are discussed in relation to the results of several recent observational studies. It is argued that all of the effects reported to date which are ascribed to the y component of the IMF can be understood, at least qualitatively, in terms of a simple theoretical picture in which the effects result from the stresses exerted on the magnetosphere consequent on the interconnection of terrestrial and interplanetary fields. In particular, relaxation under the action of these stresses allows, in effect, a partial penetration of the IMF into the magnetospheric cavity, such that the sense of the expected asymmetry effects on closed field lines can be understood, to zeroth order, in terms of the “dipole plus uniform field” model. In particular, in response to IMF By, the dayside cusp should be displaced in longitude about noon in the same sense as By in the northern hemisphere, and in the opposite sense to By in the southern hemisphere, while simultaneously the auroral oval as a whole should be shifted in the dawn-dusk direction in the opposite sense with respect to By. These expected displacements are found to be consistent with recently published observations. Similar considerations lead to the suggestion that the auroral oval may also undergo displacements in the noon-midnight direction which are associated with the x component of the IMF. We show that a previously published study of the position of the auroral oval contains strong initial evidence for the existence of this effect. However, recent results on variations in the latitude of the cusp are more ambiguous. This topic therefore requires further study before definitive conclusions can be drawn.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

The DYMECS project: a statistical approach for the evaluation of convective storms in high-resolution NWP models

A new frontier in weather forecasting is emerging by operational forecast models now being run at convection-permitting resolutions at many national weather services. However, this is not a panacea; significant systematic errors remain in the character of convective storms and rainfall distributions. The DYMECS project (Dynamical and Microphysical Evolution of Convective Storms) is taking a fundamentally new approach to evaluate and improve such models: rather than relying on a limited number of cases, which may not be representative, we have gathered a large database of 3D storm structures on 40 convective days using the Chilbolton radar in southern England. We have related these structures to storm life-cycles derived by tracking features in the rainfall from the UK radar network, and compared them statistically to storm structures in the Met Office model, which we ran at horizontal grid length between 1.5 km and 100 m, including simulations with different subgrid mixing length. We also evaluated the scale and intensity of convective updrafts using a new radar technique. We find that the horizontal size of simulated convective storms and the updrafts within them is much too large at 1.5-km resolution, such that the convective mass flux of individual updrafts can be too large by an order of magnitude. The scale of precipitation cores and updrafts decreases steadily with decreasing grid lengths, as does the typical storm lifetime. The 200-m grid-length simulation with standard mixing length performs best over all diagnostics, although a greater mixing length improves the representation of deep convective storms.

Analysis and manipulation of the structure of odor plumes from a piezo-electric release system and measurements of upwind flight of male Almond Moths, Cadra cautella, to pheromone plumes

We investigated the plume structure of a piezo-electric sprayer system, set up to release ethanol in a wind tunnel, using a fast response mini-photoionizaton detector. We recorded the plume structure of four different piezo-sprayer configurations: the sprayer alone; with a 1.6-mm steel mesh shield; with a 3.2-mm steel mesh shield; and with a 5 cm circular upwind baffle. We measured a 12 × 12-mm core at the center of the plume, and both a horizontal and vertical cross-section of the plume, all at 100-, 200-, and 400-mm downwind of the odor source. Significant differences in plume structure were found among all configurations in terms of conditional relative mean concentration, intermittency, ratio of peak concentration to conditional mean concentration, and cross-sectional area of the plume. We then measured the flight responses of the almond moth, Cadra cautella, to odor plumes generated with the sprayer alone, and with the upwind baffle piezo-sprayer configuration, releasing a 13:1 ratio of (9Z,12E)-tetradecadienyl acetate and (Z)-9-tetradecenyl acetate diluted in ethanol at release rates of 1, 10, 100, and 1,000 pg/min. For each configuration, differences in pheromone release rate resulted in significant differences in the proportions of moths performing oriented flight and landing behaviors. Additionally, there were apparent differences in the moths’ behaviors between the two sprayer configurations, although this requires confirmation with further experiments. This study provides evidence that both pheromone concentration and plume structure affect moth orientation behavior and demonstrates that care is needed when setting up experiments that use a piezo-electric release system to ensure the optimal conditions for behavioral observations.

New self-assembling multifunctional templates for the biofabrication and controlled self-release of cultured tissue

The need to source live human tissues for research and clinical applications has been a major driving force for the development of new biomaterials. Ideally, these should elicit the formation of scaffold-free tissues with native-like structure and composition. In this study, we describe a biologically interactive coating that combines the fabrication and subsequent self-release of live purposeful tissues using template–cell–environment feedback. This smart coating was formed from a self-assembling peptide amphiphile comprising a proteasecleavable sequence contiguous with a cell attachment and signaling motif. This multifunctional material was subsequently used not only to instruct human corneal or skin fibroblasts to adhere and deposit discreet multiple layers of native extracellular matrix but also to govern their own self-directed release from the template solely through the action of endogenous metalloproteases. Tissues recovered through this physiologically relevant process were carrier-free and structurally and phenotypically equivalent to their natural counterparts. This technology contributes to a new paradigm in regenerative medicine, whereby materials are able to actively direct and respond to cell behavior. The novel application of such materials as a coating capable of directing the formation and detachment of complex tissues solely under physiological conditions can have broad use for fundamental research and in future cell and tissue therapies.

Convective updraught evaluation in high-resolution NWP simulations using single-Doppler radar measurements

This study presents an evaluation of the size and strength of convective updraughts in high-resolution simulations by the UK Met Office Unified Model (UM). Updraught velocities have been estimated from range–height indicator (RHI) Doppler velocity measurements using the Chilbolton advanced meteorological radar, as part of the Dynamical and Microphysical Evolution of Convective Storms (DYMECS) project. Based on mass continuity and the vertical integration of the observed radial convergence, vertical velocities tend to be underestimated for convective clouds due to the undetected cross-radial convergence. Velocity fields from the UM at a resolution corresponding to the radar observations are used to scale such estimates to mitigate the inherent biases. The analysis of more than 100 observed and simulated storms indicates that the horizontal scale of updraughts in simulations tend to decrease with grid length; the 200 m grid length agreed most closely with the observations. Typical updraught mass fluxes in the 500 m grid length simulations were up to an order of magnitude greater than observed, and greater still in the 1.5 km grid length simulations. The effect of increasing the mixing length in the sub-grid turbulence scheme depends on the grid length. For the 1.5 km simulations, updraughts were weakened though their horizontal scale remained largely unchanged. Progressively more so for the sub-kilometre grid lengths, updraughts were broadened and intensified; horizontal scale was now determined by the mixing length rather than the grid length. In general, simulated updraughts were found to weaken too quickly with height. The findings were supported by the analysis of the widths of reflectivity patterns in both the simulations and observations.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.