Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [ nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

Viral nucleolar localisation signals determine dynamic trafficking within the nucleolus

Localisation of both viral and cellular proteins to the nucleolus is determined by a variety of factors including nucleolar localisation signals (NoLSs), but how these signals operate is not clearly understood. The nucleolar trafficking of wild type viral proteins and chimeric proteins, which contain altered NoLSs, were compared to investigate the role of NoLSs in dynamic nucleolar trafficking. Three viral proteins from diverse viruses were selected which localised to the nucleolus; the coronavirus infectious bronchitis virus nucleocapsid (N) protein, the herpesvirus saimiri ORF57 protein and the HIV-1 Rev protein. The chimeric proteins were N protein and ORF57 protein which had their own NoLS replaced with those from ORF57 and Rev proteins, respectively. By analysing the sub-cellular localisation and trafficking of these viral proteins and their chimeras within and between nucleoli using confocal microscopy and photo-bleaching we show that NoLSs are responsible for different nucleolar localisations and trafficking rates.

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

The genetic basis of resistance to anticoagulants in rodents

Anticoagulant compounds, i.e., derivatives of either 4-hydroxycoumarin (e.g., warfarin, bromadiolone) or indane-1,3-dione (e.g., diphacinone, chlorophacinone), have been in worldwide use as rodenticides for > 50 years. These compounds inhibit blood coagulation by repression of the vitamin K reductase reaction (VKOR). Anticoagulant-resistant rodent populations have been reported from many countries and pose a considerable problem for pest control. Resistance is transmitted as an autosomal dominant trait although, until recently, the basic genetic mutation was unknown. Here, we report on the identification of eight different mutations in the VKORC1 gene in resistant laboratory strains of brown rats and house mice and in wild-caught brown rats from various locations in Europe with five of these mutations affecting only two amino acids (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln, Leu128Ser). By recombinant expression of VKORC1 constructs in HEK293 cells we demonstrate that mutations at Tyr139 confer resistance to warlarin at variable degrees while the other mutations, in addition, dramatically reduce VKOR activity. Our data strongly argue for at least seven independent mutation events in brown rats and two in mice. They suggest that mutations in VKORC1 are the genetic basis of anticoagulant resistance in wild populations of rodents, although the mutations alone do not explain all aspects of resistance that have been reported. We hypothesize that these mutations, apart from generating structural changes in the VKORC1 protein, may induce compensatory mechanisms to maintain blood clotting. Our findings provide the basis for a DNA-based field monitoring of anticoagulant resistance in rodents.

DNA interaction and phosphotransfer of the C-4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Doctoral supervision of colleagues: peeling off the veneer of satisfaction and competence

The nexus of teaching, administration and research activities engaged in by academic staff in higher education means that each individual plays a multitude of roles in relation to colleagues, producing inevitable tensions. One role relationship that epitomizes this extraordinary juggling task is that of doctoral supervisor/supervisee, when both are academic staff in the same institution. The last 10 years has seen an upsurge of research interest in doctoral research students, and government and funding bodies have imposed ever more stringent requirements on supervisors. However, staff pairings have been ignored in this literature and research. This article reports on an exploration that seeks to redress this omission by giving voice to the participants (colleague supervisor or research student), allowing them to articulate constructs about what may be unique in the interaction, thus highlighting factors that support or impede success in the enterprise.

Polymer-drug conjugates: current status and future trends

Polymer conjugates are nano-sized, multicomponent constructs already in the clinic as anticancer compounds, both as single agents or as elements of combinations. They have the potential to improve pharmacological therapy of a variety of solid tumors. Polymer-drug conjugation promotes passive tumor targeting by the enhanced permeability and retention (EPR) effect and allows for lysosomotropic drug delivery following endocytic capture. In the first part of this review, we analyze the promising results arising from clinical trials of polymer-bound chemotherapy. The experience gained on these studies provides the basis for the development of a more sophisticated second-generation of polymer conjugates. However, many challenges still lay ahead providing scope to develop and refine this field. The "technology platform'' of polymer therapeutics allows the development of both new and exciting polymeric materials, the incorporation of novel bioactive agents and combinations thereof to address recent advances in drug therapy. The rational design of polymer drug conjugates is expected to realize the true potential of these "nanomedicines".

Doctoral supervision of colleagues: peeling off the veneer of satisfaction and competence

The nexus of teaching, administration and research activities engaged in by academic staff in higher education means that each individual plays a multitude of roles in relation to colleagues, producing inevitable tensions. One role relationship that epitomizes this extraordinary juggling task is that of doctoral supervisor/supervisee, when both are academic staff in the same institution. The last 10 years has seen an upsurge of research interest in doctoral research students, and government and funding bodies have imposed ever more stringent requirements on supervisors. However, staff pairings have been ignored in this literature and research. This article reports on an exploration that seeks to redress this omission by giving voice to the participants (colleague supervisor or research student), allowing them to articulate constructs about what may be unique in the interaction, thus highlighting factors that support or impede success in the enterprise.

The roles of persistence and perseveration in psychopathology

Two constructs were hypothesized to be of importance in psychological disorders: persistence (the ability to keep going to reach a goal, even when the task is difficult or drawn out) and perseveration (the tendency to continue a behavior, even when it ceases to be effective or rewarding). These are contrasted with perfectionism (having high standards for oneself or others). A measure was developed to address these constructs (the Persistence, Perseveration and Perfectionism Questionnaire; PPPQ). The PPPQ was administered to 325 nonclinical participants, alongside a measure of psychological disturbance. Factor analysis resulted in a 22-item version of the measure, consisting of subscales with good psychometric properties corresponding to the 3 theoretical constructs. Persistence was associated with lower levels of psychopathology, whereas perseveration was associated with higher levels of psychopathology. Perfectionism was weakly associated with psychopathology. Further work is needed to develop these constructs with clinical groups, but the findings support the hypothesis that persistence is an adaptive construct whereas perseveration is maladaptive.

Workflow data exchange semantics, object discovery & integration architecture for cross media production & distribution

There has been a clear lack of common data exchange semantics for inter-organisational workflow management systems where the research has mainly focused on technical issues rather than language constructs. This paper presents the neutral data exchanges semantics required for the workflow integration within the AXAEDIS framework and presents the mechanism for object discovery from the object repository where little or no knowledge about the object is available. The paper also presents workflow independent integration architecture with the AXAEDIS Framework.

Landscape of intelligent cores: An autonomic multi-agent approach for space application

Space applications are challenged by the reliability of parallel computing systems (FPGAs) employed in space crafts due to Single-Event Upsets. The work reported in this paper aims to achieve self-managing systems which are reliable for space applications by applying autonomic computing constructs to parallel computing systems. A novel technique, 'Swarm-Array Computing' inspired by swarm robotics, and built on the foundations of autonomic and parallel computing is proposed as a path to achieve autonomy. The constitution of swarm-array computing comprising for constituents, namely the computing system, the problem / task, the swarm and the landscape is considered. Three approaches that bind these constituents together are proposed. The feasibility of one among the three proposed approaches is validated on the SeSAm multi-agent simulator and landscapes representing the computing space and problem are generated using the MATLAB.

Towards self-ware via swarm-array computing

The work reported in this paper proposes Swarm-Array computing, a novel technique inspired by swarm robotics, and built on the foundations of autonomic and parallel computing. The approach aims to apply autonomic computing constructs to parallel computing systems and in effect achieve the self-ware objectives that describe self-managing systems. The constitution of swarm-array computing comprising four constituents, namely the computing system, the problem/task, the swarm and the landscape is considered. Approaches that bind these constituents together are proposed. Space applications employing FPGAs are identified as a potential area for applying swarm-array computing for building reliable systems. The feasibility of a proposed approach is validated on the SeSAm multi-agent simulator and landscapes are generated using the MATLAB toolkit.

The risk-taking and self-harm inventory for adolescents: Development and psychometric evaluation

In this study, we report on the development and psychometric evaluation of the Risk-Taking (RT) and Self-Harm (SH) Inventory for Adolescents (RTSHIA), a self-report measure designed to assess adolescent RT and SH in community and clinical settings. 651 young people from secondary schools in England ranging in age from 11.6 years to 18.7 years and 71 young people referred to mental health services for SH behavior in London between the ages of 11.9 years and 17.5 years completed the RTSHIA along with standardized measures of adolescent psychopathology. Two factors emerged from the principal axis factoring, and RT and SH were further validated by a confirmatory factor analysis as related, but different, constructs, rather than elements of a single continuum. Inter-item and test–retest reliabilities were high for both components (Cronbach's α = .85, rtt = .90; Cronbach's α .93, rtt = .87), and considerable evidence emerged in support of the measure's convergent, concurrent, and divergent validity. The findings are discussed with regard to potential usefulness of the RTSHIA for research and clinical purposes with adolescents.

Modula-2 applied

Programming is a skill which requires knowledge of both the basic constructs of the computer language used and techniques employing these constructs. How these are used in any given application is determined intuitively, and this intuition is based on experience of programs already written. One aim of this book is to describe the techniques and give practical examples of the techniques in action - to provide some experience. Another aim of the book is to show how a program should be developed, in particular how a relatively large program should be tackled in a structured manner. These aims are accomplished essentially by describing the writing of one large program, a diagram generator package, in which a number of useful programming techniques are employed. Also, the book provides a useful program, with an in-built manual describing not only how the program works, but also how it does it, with full source code listings. This means that the user can, if required, modify the package to meet particular requirements. A floppy disk is available from the publishers containing the program, including listings of the source code. All the programs are written in Modula-2, using JPI's Top Speed Modula-2 system running on IBM-PCs and compatibles. This language was chosen as it is an ideal language for implementing large programs and it is the main language taught in the Cybernetics Department at the University of Reading. There are some aspects of the Top Speed implementation which are not standard, so suitable comments are given when these occur. Although implemented in Modula-2, many of the techniques described here are appropriate to other languages, like Pascal of C, for example. The book and programs are based on a second year undergraduate course taught at Reading to Cybernetics students, entitled Algorithms and Data Structures. Useful techniques are described for the reader to use, applications where they are appropriate are recommended, but detailed analyses of the techniques are not given.

A modular head/eye platform for real-time reactive vision

This paper describes the design, implementation and testing of a high speed controlled stereo “head/eye” platform which facilitates the rapid redirection of gaze in response to visual input. It details the mechanical device, which is based around geared DC motors, and describes hardware aspects of the controller and vision system, which are implemented on a reconfigurable network of general purpose parallel processors. The servo-controller is described in detail and higher level gaze and vision constructs outlined. The paper gives performance figures gained both from mechanical tests on the platform alone, and from closed loop tests on the entire system using visual feedback from a feature detector.