COPASAAR – A database for proteomic analysis of single amino acid repeats

BACKGROUND: Single amino acid repeats make up a significant proportion in all of the proteomes that have currently been determined. They have been shown to be functionally and medically significant, and are associated with cancers and neuro-degenerative diseases such as Huntington's Chorea, where a poly-glutamine repeat is responsible for causing the disease. The COPASAAR database is a new tool to facilitate the rapid analysis of single amino acid repeats at a proteome level. The database aims to simplify the comparison of repeat distributions between proteomes in order to provide a better understanding of their function and evolution. RESULTS: A comparative analysis of all proteomes in the database (currently 244) shows that single amino acid repeats account for about 12–14% of the proteome of any given species. They are more common in eukaryotes (14%) than in either archaea or bacteria (both 13%). Individual analyses of proteomes show that long single amino acid repeats (6+ residues) are much more common in the Eukaryotes and that longer repeats are usually made up of hydrophilic amino acids such as glutamine, glutamic acid, asparagine, aspartic acid and serine. CONCLUSION: COPASAAR is a useful tool for comparative proteomics that provides rapid access to amino acid repeat data that can be readily data-mined. The COPASAAR database can be queried at the kingdom, proteome or individual protein level. As the amount of available proteome data increases this will be increasingly important in order to automate proteome comparison. The insights gained from these studies will give a better insight into the evolution of protein sequence and function

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops

Label-free segmentation of co-cultured cells on a nanotopographical gradient

The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity

Genetic testing of children for adult-onset conditions: opinions of the British adult population and implications for clinical practice

This study set out to explore the attitudes of a representative sample of the British public towards genetic testing in children to predict disease in the future. We sought opinions about genetic testing for adult-onset conditions for which no prevention/treatment is available during childhood, and about genetic 'carrier' status to assess future reproductive risks. The study also examined participants' level of agreement with the reasons professional organisations give in favour of deferring such testing. Participants (n=2998) completed a specially designed questionnaire, distributed by email. Nearly half of the sample (47%) agreed that parents should be able to test their child for adult-onset conditions, even if there is no treatment or prevention at time of testing. This runs contrary to professional guidance about genetic testing in children. Testing for carrier status was supported by a larger proportion (60%). A child's future ability to decide for her/himself if and when to be tested was the least supported argument in favour of deferring testing.European Journal of Human Genetics advance online publication, 5 November 2014; doi:10.1038/ejhg.2014.221

Novel statistical approaches for non-normal censored immunological data: analysis of cytokine and gene expression data

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets

Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs

It is emerging that nanotopographical information can be used to induce osteogenesis from mesenchymal stromal cells from the bone marrow and it is hoped that this nanoscale bioactivity can be utilized to engineer next generation implants. However, the osteogenic mechanism of surfaces is currently poorly understood. In this report, we investigate mechanism and implicate bone morphogenic protein (BMP) in up-regulation of RUNX2 and show that RUNX2 and its regulatory miRNAs are BMP sensitive. Our data demonstrates that osteogenic nanotopography promotes co-localization of intergrins and BMP2 receptors in order to enhance osteogenic activity and that vitronectin is important in this interface. This provides insight that topographical regulation of adhesion can have effects on signaling cascades outside of cytoskeletal signaling and that adhesions can have roles in augmenting BMP signaling

The environmental security debate and its significance for climate change

Policymakers, military strategists and academics all increasingly hail climate change as a security issue. This article revisits the (comparatively) long-standing “environmental security debate” and asks what lessons that earlier debate holds for the push towards making climate change a security issue. Two important claims are made. First, the emerging climate security debate is in many ways a re-run of the earlier dispute. It features many of the same proponents and many of the same disagreements. These disagreements concern, amongst other things, the nature of the threat, the referent object of security and the appropriate policy responses. Second, given its many different interpretations, from an environmentalist perspective, securitisation of the climate is not necessarily a positive development

Applications of Direct Injection Soft Chemical Ionisation-Mass Spectrometry for the Detection of Pre-blast Smokeless Powder Organic Additives

Analysis of smokeless powders is of interest from forensics and security perspectives. This article reports the detection of smokeless powder organic additives (in their pre-detonation condition), namely the stabiliser diphenylamine and its derivatives 2-nitrodiphenylamine and 4-nitrodiphenylamine, and the additives (used both as stabilisers and plasticisers) methyl centralite and ethyl centralite, by means of swab sampling followed by thermal desorption and direct injection soft chemical ionisation-mass spectrometry. Investigations on the product ions resulting from the reactions of the reagent ions H3O+ and O2+ with additives as a function of reduced electric field are reported. The method was comprehensively evaluated in terms of linearity, sensitivity and precision. For H3O+, the limits of detection (LoD) are in the range of 41-88 pg of additive, for which the accuracy varied between 1.5 and 3.2%, precision varied between 3.7 and 7.3% and linearity showed R20.9991. For O2+, LoD are in the range of 72 to 1.4 ng, with an accuracy of between 2.8 and 4.9% and a precision between 4.5 and 8.6% and R20.9914. The validated methodology was applied to the analysis of commercial pre-blast gun powders from different manufacturers.(VLID)4826148Accepted versio

Negotiating the inhuman: Bakhtin, materiality and the instrumentalization of climate change

The article argues that the work of literary theorist Mikhail M. Bakhtin presents a starting point for thinking about the instrumentalization of climate change. Bakhtin’s conceptualization of human–world relationships, encapsulated in the concept of ‘cosmic terror’, places a strong focus on our perception of the ‘inhuman’. Suggesting a link between the perceived alienness and instability of the world and in the exploitation of the resulting fear of change by political and religious forces, Bakhtin asserts that the latter can only be resisted if our desire for a false stability in the world is overcome. The key to this overcoming of fear, for him, lies in recognizing and confronting the worldly relations of the human body. This consciousness represents the beginning of one’s ‘deautomatization’ from following established patterns of reactions to predicted or real changes. In the vein of several theorists and artists of his time who explored similar ‘deautomatization’ strategies – examples include Shklovsky’s ‘ostranenie’, Brecht’s ‘Verfremdung’, Artaud’s emotional ‘cruelty’ and Bataille’s ‘base materialism’ – Bakhtin proposes a more playful and widely accessible experimentation to deconstruct our ‘habitual picture of the world’. Experimentation is envisioned to take place across the material and the textual to increase possibilities for action. Through engaging with Bakhtin’s ideas, this article seeks to draw attention to relations between the imagination of the world and political agency, and the need to include these relations in our own experiments with creating climate change awareness