Structural characterisation of hypoxia-mimicking bioactive glasses

Nickel and cobalt are both known to stimulate the hypoxia-inducible factor-1 (HIF-1a), thus significantly improving blood vessel formation in tissue engineering applications. We have manufactured nickel and cobalt doped bioactive glasses to act as a controlled delivery mechanism of these ions. The resultant structural consequences have been investigated using the methods of isotopic and isomorphic substitution applied to neutron diffraction. The structural sites present will be intimately related to their release properties in physiological fluids such as plasma and saliva, and hence the bioactivity of the material. Detailed structural knowledge is therefore a prerequisite for optimising material design. Results show that nickel and cobalt adopt a mixed structural role within these bioactive glasses occupying both network-forming (tetrahedral) and network-modifying (5-fold) geometries. Two thirds of the Ni (or Co) occupies a five-fold geometry with the remaining third in a tetrahedral environment. A direct comparison of the primary structural correlations (e.g. Si-O, Ca-O, Na-O and O-Si-O) between the archetypal 45S5 Bioglass® and the Ni and Co glasses studied here reveal no significant differences. This indicates that the addition of Ni (or Co) will have no adverse effects on the existing structure, and thus on in vitro/in vivo dissolution rates and therefore bioactivity of these glasses.

Structural effects in photopolymerized sodium AMPS hydrogels crosslinked with poly(ethylene glycol) diacrylate for use as burn dressings

Synthetic hydrogel polymers were prepared by free radical photopolymerization in aqueous solution of the sodium salt of 2-acrylamido-2-methylpropane sulfonic acid (Na-AMPS). Poly(ethylene glycol) diacrylate (PEGDA) and 4,4'-azo-bis(4-cyanopentanoic acid) were used as the crosslinker and UV-photoinitiator, respectively. The effects of varying the Na-AMPS monomer concentration within the range of 30-50% w/v and the crosslinker concentration within the range of 0.1-1.0% mol (relative to monomer) were studied in terms of their influence on water absorption properties. The hydrogel sheets exhibited extremely high swelling capacities in aqueous media which were dependent on monomer concentration, crosslink density, and the ionic strength and composition of the immersion medium. The effects of varying the number-average molecular weight of the PEGDA crosslinker from = 250 to 700 were also investigated. Interestingly, it was found that increasing the molecular weight and therefore the crosslink length at constant crosslink density decreased both the rate of water absorption and the equilibrium water content. Cytotoxicity testing by the direct contact method with mouse fibroblast L929 cells indicated that the synthesized hydrogels were nontoxic. On the basis of these results, it is considered that photopolymerized Na-AMPS hydrogels crosslinked with PEGDA show considerable potential for biomedical use as dressings for partial thickness burns. This paper describes some structural effects which are relevant to their design as biomaterials for this particular application. © 2013 Copyright Taylor and Francis Group, LLC.

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10 molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies-characterized by a fixed number of paired nucleotides-and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited. © 2008 Elsevier Ltd. All rights reserved.

Structural optimization

The non-linear programming algorithms for the minimum weight design of structural frames are presented in this thesis. The first, which is applied to rigidly jointed and pin jointed plane frames subject to deflexion constraints, consists of a search in a feasible design space. Successive trial designs are developed so that the feasibility and the optimality of the designs are improved simultaneously. It is found that this method is restricted lo the design of structures with few unknown variables. The second non-linear programming algorithm is presented .in a general form. This consists of two types of search, one improving feasibility and the other optimality. The method speeds up the 'feasible direction' approach by obtaining a constant weight direction vector that is influenced by dominating constraints. For pin jointed plane and space frames this method is used to obtain a 'minimum weight' design which satisfies restrictions on stresses and deflexions. The matrix force method enables the design requirements to be expressed in a general form and the design problem is automatically formulated within the computer. Examples are given to explain the method and the design criteria are extended to include member buckling. Fundamental theorems are proposed and proved to confirm that structures are inter-related. These theorems are applicable to linear elastic structures and facilitate the prediction of the behaviour of one structure from the results of analysing another, more general, or related structure. It becomes possible to evaluate the significance of each member in the behaviour of a structure and the problem of minimum weight design is extended to include shape. A method is proposed to design structures of optimum shape with stress and deflexion limitations. Finally a detailed investigation is carried out into the design of structures to study the factors that influence their shape.

Senior management leadership, social support, job design and stressor-to-strain relationships in hospital practice

Purpose: The purpose of this paper is to examine the effect of the quality of senior management leadership on social support and job design, whose main effects on strains, and moderating effects on work stressors-to-strains relationships were assessed. Design/methodology/approach: A survey involving distribution of questionnaires was carried out on a random sample of health care employees in acute hospital practice in the UK. The sample comprised 65,142 respondents. The work stressors tested were quantitative overload and hostile environment, whereas strains were measured through job satisfaction and turnover intentions. Structural equation modelling and moderated regression analyses were used in the analysis. Findings: Quality of senior management leadership explained 75 per cent and 94 per cent of the variance of social support and job design respectively, whereas work stressors explained 51 per cent of the variance of strains. Social support and job design predicted job satisfaction and turnover intentions, as well as moderated significantly the relationships between quantitative workload/hostility and job satisfaction/turnover intentions. Research limitations/implications: The findings are useful to management and to health employees working in acute/specialist hospitals. Further research could be done in other counties to take into account cultural differences and variations in health systems. The limitations included self-reported data and percept-percept bias due to same source data collection. Practical implications: The quality of senior management leaders in hospitals has an impact on the social environment, the support given to health employees, their job design, as well as work stressors and strains perceived. Originality/value: The study argues in favour of effective senior management leadership of hospitals, as well as ensuring adequate support structures and job design. The findings may be useful to health policy makers and human resources managers. © Emerald Group Publishing Limited.

Using genetic programming and decision trees for generating structural descriptions of four bar mechanisms

Four bar mechanisms are basic components of many important mechanical devices. The kinematic synthesis of four bar mechanisms is a difficult design problem. A novel method that combines the genetic programming and decision tree learning methods is presented. We give a structural description for the class of mechanisms that produce desired coupler curves. Constructive induction is used to find and characterize feasible regions of the design space. Decision trees constitute the learning engine, and the new features are created by genetic programming.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.