Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Value management and post-occupancy evaluation: closing the loop

This paper considers the relationship between value management and facilities management. The findings are particularly relevant to large client organisations which procure new buildings on a regular basis. It is argued that the maximum effectiveness of value management can only be achieved if it is used in conjunction with an ongoing commitment to post-occupancy evaluation. SMART value management is seen to provide the means of ensuring that an individual building design is in alignment with the client’s strategic property needs. However, it is also necessary to recognise that an organisation’s strategic property needs will continually be in a state of change. Consequentially, economic and functional under-performance can only be avoided by a regular performance audit of existing property stock in accordance with changing requirements. Such a policy will ensure ongoing competitiveness through organisational learning. While post-occupancy evaluation represents an obvious additional service to be provided by value management consultants, it is vital that the necessary additional skills are acquired. Process management skills and social science research techniques are clearly important. However, there is also a need to improve mechanisms for data manipulation. Success can only be achieved if equal attention is given to issues of process, structure and content.

Self-assembly of Fmoc-tetrapeptides based on the RGDS cell adhesion motif

Self-assembly in aqueous solution has been investigated for two Fmoc [Fmoc ¼ N-(fluorenyl)-9-methoxycarbonyl] tetrapeptides comprising the RGDS cell adhesion motif from fibronectin or the scrambled sequence GRDS. The hydrophobic Fmoc unit confers amphiphilicity on the molecules, and introduces aromatic stacking interactions. Circular dichroism and FTIR spectroscopy show that the self-assembly of both peptides at low concentration is dominated by interactions among Fmoc units, although Fmoc-GRDS shows b-sheet features, at lower concentration than Fmoc-RGDS. Fibre X-ray diffraction indicates b-sheet formation by both peptides at sufficiently high concentration. Strong alignment effects are revealed by linear dichroism experiments for Fmoc-GRDS. Cryo-TEM and smallangle X-ray scattering (SAXS) reveal that both samples form fibrils with a diameter of approximately 10 nm. Both Fmoc-tetrapeptides form self-supporting hydrogels at sufficiently high concentration. Dynamic shear rheometry enabled measurements of the moduli for the Fmoc-GRDS hydrogel, however syneresis was observed for the Fmoc-RGDS hydrogel which was significantly less stable to shear. Molecular dynamics computer simulations were carried out considering parallel and antiparallel b-sheet configurations of systems containing 7 and 21 molecules of Fmoc-RGDS or Fmoc-GRDS, the results being analyzed in terms of both intermolecular structural parameters and energy contributions.

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A–B-loop mimetics of the Cε3 domain of human IgE

The disruption of the human immunolobulin E–high affinity receptor I (IgE–FcεRI) protein–protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A–B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC50 660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A–B loop epitope in IgE.

Closed loop design for a simple nonlinear aircraft model using eigenstructure assignment

Aircraft systems are highly nonlinear and time varying. High-performance aircraft at high angles of incidence experience undesired coupling of the lateral and longitudinal variables, resulting in departure from normal controlled flight. The aim of this work is to construct a robust closed-loop control that optimally extends the stable and decoupled flight envelope. For the study of these systems nonlinear analysis methods are needed. Previously, bifurcation techniques have been used mainly to analyze open-loop nonlinear aircraft models and investigate control effects on dynamic behavior. In this work linear feedback control designs calculated by eigenstructure assignment methods are investigated for a simple aircraft model at a fixed flight condition. Bifurcation analysis in conjunction with linear control design methods is shown to aid control law design for the nonlinear system.

L'intégration littéraire et iconographique du motif de la descente du Christ aux Enfers à l'ouverture du Merlin

Le début du Merlin et sa mise en image témoignent de l’influence paradoxale du matériau biblique et apocryphe sur la littérature arthurienne, dite "profane". A travers les représentations de la descente aux enfers et le récit de l'effort avorté de conception d'un antéchrist, le roman se joue du texte sacré et de la tradition auxquels il emprunte sa matière. Dans le contexte médiéval, cette réécriture correspond moins à une entreprise de désacralisation et de transgression qu’à la libre continuation du mouvement de réflexion et de création suscité par la matière biblique.

Comparing the thermal performance of horizontal slinky-loop and vertical slinky-loop heat exchangers

The heat pump market in the UK has grown rapidly over the last few years. Performance analyses of vertical ground-loop heat exchanger configurations have been widely carried out using both numerical modelling and experiments. However, research findings and design recommendations on horizontal slinky-loop and vertical slinky-loop heat exchangers are far fewer compared with those for vertical ground-loop heat exchanger configurations, especially where the long-term operation of the systems is concerned. The paper presents the results obtained from a numerical simulation for the horizontal slinky-loop and vertical slinky-loop heat exchangers of a ground-source heat pump system. A three-dimensional numerical heat transfer model was developed to study the thermal performance of various heat exchanger configurations. The influence of the loop pitch (loop spacing) and the depth of a vertical slinky-loop installation were investigated and the thermal performance and excavation work required for the horizontal and vertical slinky-loop heat exchangers were compared. The influence of the installation depth for vertical slinky-loop configurations was also investigated. The results of this study show that the influence of the installation depth of the vertical slinky-loop heat exchanger on the thermal performance of the system is small. The maximum difference in the thermal performance between the vertical and horizontal slinky-loop heat exchangers with the same loop diameter and loop pitch is less than 5%.

Simulation of thermal performance of horizontal slinky-loop heat exchangers for ground source heat pumps

This paper presents results obtained from a numerical simulation for the horizontal slinky-loop heat exchanger of a ground-source heat pump system. A three-dimensional numerical model was developed and the results of the thermal performance of various heat exchanger configurations are presented. The investigation was carried out on five types of loop pitch (loop spacing), three types of loop diameter, three values of soil thermal properties, and allowing continuous and intermittent operation. Comparison was made for the heat transfer rate, the amount of pipe material needed, as well as excavation work required for the horizontal slinky-loop heat exchanger. The results indicate that system parameters have a significant effect on the thermal performance of the system

Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signalling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Solar origin of heliospheric magnetic field inversions: evidence for coronal loop opening within pseudostreamers

The orientation of the heliospheric magnetic field (HMF) in near‒Earth space is generally a good indicator of the polarity of HMF foot points at the photosphere. There are times, however, when the HMF folds back on itself (is inverted), as indicated by suprathermal electrons locally moving sunward, even though they must ultimately be carrying the heat flux away from the Sun. Analysis of the near‒Earth solar wind during the period 1998–2011 reveals that inverted HMF is present approximately 5.5% of the time and is generally associated with slow, dense solar wind and relatively weak HMF intensity. Inverted HMF is mapped to the coronal source surface, where a new method is used to estimate coronal structure from the potential‒field source‒surface model. We find a strong association with bipolar streamers containing the heliospheric current sheet, as expected, but also with unipolar or pseudostreamers, which contain no current sheet. Because large‒scale inverted HMF is a widely accepted signature of interchange reconnection at the Sun, this finding provides strong evidence for models of the slow solar wind which involve coronal loop opening by reconnection within pseudostreamer belts as well as the bipolar streamer belt. Occurrence rates of bipolar‒ and pseudostreamers suggest that they are equally likely to result in inverted HMF and, therefore, presumably undergo interchange reconnection at approximately the same rate. Given the different magnetic topologies involved, this suggests the rate of reconnection is set externally, possibly by the differential rotation rate which governs the circulation of open solar flux.