Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome

Different routes, common directions? Activation policies for young people in Denmark and the UK

This article analyses and compares the development of activation policies for young people in Denmark and the UK from the mid-1990s. Despite their diverse welfare traditions and important differences in the organisation and delivery of benefits and services for the unemployed, both countries have recently introduced large-scale compulsory activation programmes for young people. These programmes share a number of common features, especially a combination of strong compulsion and an apparently contradictory emphasis on client-centred training and support for participants. The suggested transition from the ‘Keynesian welfare state’ to the ‘Schumpeterian workfare regime’ is used as a framework to discuss the two countries’ recent moves towards activation. It is argued that while this framework is useful in explaining the general shift towards active labour-market policies in Europe, it alone cannot account for the particular convergence of the Danish and British policies in the specific area of youth activation. Rather, a number of specific political factors explaining the development of policies in the mid-1990s are suggested. The article concludes that concerns about mass youth unemployment, the influence of the ‘dependency culture’ debate in various forms, cross-national policy diffusion and, crucially, the progressive re-engineering of compulsory activation by strong centre-left governments have all contributed to the emergence of policies that mix compulsion and a commitment to the centrality of work with a ‘client-centred approach’ that seeks to balance more effective job seeking with human resource development. However, attempts to combine the apparently contradictory concepts of ‘client-centredness’ and compulsion are likely to prove politically fragile, and both countries risk lurching towards an increasingly workfarist approach

Loss firms' annual report narratives and share price anticipation of earnings

We extend prior research into the association between disclosure quality and share price anticipation of earnings by discriminating between firms that report profits and firms that report losses. As a measure of disclosure quality we count the number of forward-looking profit statements in annual report narratives. To measure the extent to which current share price movements anticipate future earnings changes we regress current stock returns on current and future earnings changes. The coefficients on the future earnings change variables are our measure of share price anticipation of earnings. Our regression results show that the association between annual report narratives and share price anticipation of earnings is not the same for profit and loss firms. For loss firms we find that the ability of stock returns to anticipate next period’s earnings change is significantly greater when the firm provides a large number of profit predictions in annual report narratives. We make no such observation for profit firms. In addition, once we control for variations in the intrinsic lead-lag relation between returns and earnings across industries, the observed difference between profit and loss firms becomes statistically significant. Overall, our results are consistent with annual report narratives being a particularly important source of information for loss-making firms

Untangling the Causal Effects of Sex on Judging

We explore the role of sex in judging by addressing two questions of long-standing interest to political scientists: whether and in what ways male and female judges decide cases distinctly—“individual effects”—and whether and in what ways serving with a female judge causes males to behave differently—“panel effects.” While we attend to the dominant theoretical accounts of why we might expect to observe either or both effects, we do not use the predominant statistical tools to assess them. Instead, we deploy a more appropriate methodology: semiparametric matching, which follows from a formal framework for causal inference. Applying matching methods to 13 areas of law, we observe consistent gender effects in only one—sex discrimination. For these disputes, the probability of a judge deciding in favor of the party alleging discrimination decreases by about 10 percentage points when the judge is a male. Likewise, when a woman serves on a panel with men, the men are significantly more likely to rule in favor of the rights litigant. These results are consistent with an informational account of gendered judging and are inconsistent with several othersPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116101/1/ajps10.pd

Police accountability and the Irish law of evidence

"Common law courts have differed on whether and to what extent an exclusionary rule should be used as a tool to impose standards on the police. The Irish courts have pursued an uncompromising approach in this area. Basing themselves on the imperative of upholding the constitutional rights of the accused, they have been willing to exclude relevant and cogent evidence on the basis that it was obtained by the police in breach of those rights. This article locates the Irish constitutional exclusionary rule in the broader context of the role of the law of evidence in police governance. Citing specific examples from the Irish legislation and case law, it shows how recent legislative interventions and some judicial hesitancy have fuelled inconsistent and contradictory trends. It concludes that there is now a pressing need for reflection on the respective roles of the legislature and the courts in this area." [author's abstract

The complexity of kidney disease and diagnosing it – cystatin C, selective glomerular hypofiltration syndromes and proteome regulation

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5–30 kDa molecules, such as cystatin C, compared to the filtration of small molecules <1 kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.publishedVersio

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein–protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in ‘loop 2’ allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression

Activation of natural regulatory T cells by IgG Fc-derived peptide Tregitopes

We have identified at least 2 highly promiscuous major histocompatibility complex class II T-cell epitopes in the Fc fragment of IgG that are capable of specifically activating CD4+CD25HiFoxP3+ natural regulatory T cells (nTRegs). Coincubation of these regulatory T-cell epitopes or “Tregitopes” and antigens with peripheral blood mononuclear cells led to a suppression of effector cytokine secretion, reduced proliferation of effector T cells, and caused an increase in cell surface markers associated with TRegs such as FoxP3. In vivo administration of the murine homologue of the Fc region Tregitope resulted in suppression of immune response to a known immunogen. These data suggest that one mechanism for the immunosuppressive activity of IgG, such as with IVIG, may be related to the activity of regulatory T cells. In this model, regulatory T-cell epitopes in IgG activate a subset of nTRegs that tips the resulting immune response toward tolerance rather than immunogenicity