Cellulases from extremely thermophilic bacteria

Cellulose is the most abundant biopolymer on earth, and is the major component of urban waste. Thus cellulose must be seen as a very significant renewable source of chemical foodstocks when fossil fuels become restricted

Phytochrome gene diversity

The structures and functions of the phytochrome apoprotein genes (the PHY genes), their diversity across the plant kingdom, and their evolution are central concerns in the study of red-light sensing in plants. We summarize here recent advances in two areas relating to these topics: (1) the characteristics of the PHY gene family in Arabidopsis thaliana, the higher plant species for which the most extensive information on these genes is available, and (2) the similarity relationships, phylogeny, and evolutionary implications of PHY gene sequences and partial sequences which have been described from various plants. Together, these two areas of study, one directed at understanding in detail the phytochromes present in a single species and the other directed at a much broader understanding of PHY gene relatedness and distribution, are producing an increasingly clear picture of the diversity and evolution of plant red-light photoreceptors. Moreover, they suggest that the complexity of the phytochrome family has increased as land plants have evolved novel morphologies

Family Law

Family La

Using E. coli NfsA as a model to improve our understanding of enzyme engineering

There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to the difficulty for step-wise evolutionary approaches in effectively retaining mutations that are beneficial in combination with one another, but on an individual basis are neutral or deleterious (i.e., exhibit positive epistasis). We sought to determine whether a superior enzyme identified using a simultaneous mass site directed mutagenesis approach could have been identified using a step-wise approach. We conducted simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 394 million unique variants. We then applied a powerful positive selection using chloramphenicol which is toxic to E. coli but can be detoxified via nitro-reduction. This has enabled us to recover a diverse range of highly active nitroreductase variants. For two of the most active variants, we have created all possible combinations of single mutations. This allowed us to examine whether a step-wise mutagenesis pathway could have also yielded these enzymes. As anticipated, we identified complex epistatic interactions between residues in these enzyme variants. We have also investigated the “black-box” effect of enzyme engineering, examining the consequences that evolving NfsA towards one specialist activity had on the other promiscuous activities of NfsA. Variants generated in this study have also had practical applications, in particular for targeted cell ablation in zebrafish. We have identified NfsA variants that are highly active with nil-bystander prodrugs that can selectively ablate nitroreductase expressing cells without harm to adjacent cells. In ongoing work, our lead variants are being evaluated for their utility in transgenic zebrafish models of degenerative disease

Setting a research agenda for progressive multiple sclerosis: The International Collaborative on Progressive MS

Despite significant progress in the development of therapies for relapsing MS, progressive MS remains comparatively disappointing. Our objective, in this paper, is to review the current challenges in developing therapies for progressive MS and identify key priority areas for research. A collaborative was convened by volunteer and staff leaders from several MS societies with the mission to expedite the development of effective disease-modifying and symptom management therapies for progressive forms of multiple sclerosis. Through a series of scientific and strategic planning meetings, the collaborative identified and developed new perspectives on five key priority areas for research: experimental models, identification and validation of targets and repurposing opportunities, proof-of-concept clinical trial strategies, clinical outcome measures, and symptom management and rehabilitation. Our conclusions, tackling the impediments in developing therapies for progressive MS will require an integrated, multi-disciplinary approach to enable effective translation of research into therapies for progressive MS. Engagement of the MS research community through an international effort is needed to address and fund these research priorities with the ultimate goal of expediting the development of disease-modifying and symptom-relief treatments for progressive MS

The Claims Culture: A Taxonomy of Industry Attitudes

This paper presents an analysis of a familiar aspect of construction industry culture that we have dubbed 'the claims culture'. This is a culture of contract administration that lays a strong emphasis on the planning and management of claims. The principal elements of the analysis are two sets of distinctions. The first comprises economic and occupational orders, referring to two kinds of control that are exercised over the construction process; predicated respectively on economic ownership and occupational competence. The second refers to contrasting attitudes towards relationships and problem solving within these orders: respectively 'distributive' and 'integrative'. The concepts of economic and occupational order entail further sub-categories. The various attitudes associated with these categories and sub-categories are described. They are assessed as to their consequences for change initiatives in the industry

Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall