Comparative proteomic profiling reveals mechanisms for early spinal cord vulnerability in CLN1 disease

CLN1 disease is a fatal inherited neurodegenerative lysosomal storage disease of early childhood, caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl protein thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1−/−) mice and human CLN1 disease that contributes to clinical outcome and precedes the onset of brain pathology. Here, we quantified this spinal pathology at 3 and 7 months of age revealing significant and progressive glial activation and vulnerability of spinal interneurons. Tandem mass tagged proteomic analysis of the spinal cord of Ppt1−/−and control mice at these timepoints revealed a significant neuroimmune response and changes in mitochondrial function, cell-signalling pathways and developmental processes. Comparing proteomic changes in the spinal cord and cortex at 3 months revealed many similarly affected processes, except the inflammatory response. These proteomic and pathological data from this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies. These data also fundamentally change our understanding of the progressive, site-specific nature of CLN1 disease pathogenesis, and highlight the importance of the neuroimmune response. This should greatly impact our approach to the timing and targeting of future therapeutic trials for this and similar disorders

Analysis of the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate by active-site mutants of Aspergillus nidulans isopenicillin N synthase.

BACKGROUND: Penicillins and cephalosporins constitute a major class of clinically useful antibiotics. A key step in their biosynthesis involves the oxidative cyclisation of delta-(Lalpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N by isopenicillin N synthase (IPNS). This chemically remarkable transformation has been extensively studied using substrate analogues. The conversion of an analogue in which the valine is replaced by alpha-aminobutyrate results in three products, two epimeric penams and a cepham. The ratio of these products in reactions catalysed by four different IPNS isozymes has been used previously to probe the thermicity of the chemical mechanism. But how IPNS restricts the products from the natural substrate to a single penam (isopenicillin N) has remained unknown. RESULTS: A key active-site residue, Leu223, identified according to a model of enzyme-substrate binding, has been altered to sterically less demanding residues. As the steric constraints on the upper part of the active site are reduced, the ratio of the beta-methyl penam to the cepham increases when the alpha-aminobutyrate-containing substrate analogue is used. These results suggest a mechanism for processing of the natural substrate in which IPNS uses steric control to restrict the conformational freedom of an intermediate such that the only product is the penam. CONCLUSIONS: Using steric pressure to control conformation, and hence to disfavour reactions leading to alternate products, is probably the result of evolutionary selection for a biologically active product at the expense of biologically inactive byproducts. It is likely that this sort of enzymatic catalysis is used in situations where substrate conversion is highly exothermic and a variety of products are possible

Precedence-type tests based on record values

Precedence-type tests, Records, Distribution-free tests, Lehmann alternative, Uniformly most powerful test,

A study of a technological development process: Human factors—the forgotten factors?

Author's accepted version (post-print).The final publication is available at Springer via http://dx.doi.org/10.1007/s10111-016-0379-x.Available from 31/05/2017.The aim of this study was to explore how human factors were taken into account in the development of a new type of drilling equipment. This study is part of a larger project on the understanding of human factors in the design and implementation of automated drilling technology. The principal study was a longitudinal study lasting 4 years that involved 43 interviews, offshore and onshore observations, and two surveys. The analysis in this paper is based on seven informants who were either part of the design team or the paramount project team developing new automated drilling technology for an offshore oil- and gas-producing installation in the same development project, in addition to project documents. The informants were interviewed using semi-structured interviews, and grounded theory based on the coding process of Strauss and Corbin (Basics of qualitative research: grounded theory procedures and techniques. Sage, Newbury Park, 1990) was used to analyse the data. The core category was found to be insufficient human factor analyses performed in the development phase due to the two main categories, namely (1) insufficient information coordination and (2) narrow focus in different phases of the project. This was found to contribute to increased costs, low user-friendliness, and end users’ insufficient knowledge of safe usage and potential risks. Our conclusion was that homogenous top competence involving technical aspects contributed to developers’ lack of understanding of the need for sufficient analyses of end user requirements and of the tasks that would be affected by the new technology. Hence, we argue that technological development could benefit from including human factors experts from the project’s outset to bridge the gap between the lack of relevant information and sufficient information on which to base development decisions. In addition, we contend that performing human factors analyses throughout the development of a project would be beneficial due to the potential of hindering cultural aspects such as a non-questioning culture, which is viewed as a hazard in high-risk organizations