Interrogating fragments using a protein thermal shift assay

Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand-protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein-drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein-protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD campaign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree

Backbone and side chain H-1, N-15 and C-13 assignments for the oxidised and reduced forms of the oxidoreductase protein DsbA from Staphylococcus aureus

The function and dynamics of the thiol-disulfide oxidoreductase DsbA in the low-GC gram positive bacterium, Staphylococcus aureus, are yet to be elucidated. Here we report 13C, 15N and 1H assignments for the oxidised and reduced forms of SaDsbA as a prelude to further studies on the enzyme

Digital Scholarship Considered: How New Technologies Could Transform Academic Work

New digital and web-based technologies are spurring rapid and radical changes across all media industries. These newer models take advantage of the infinite reproducibility of digital media at zero marginal cost. There is an argument to be made that the sort of changes we have seen in other industries will be forced upon higher education, either as the result of external economic factors (the need to be more efficient, responsive, etc.) or by a need to stay relevant to the so-called ‘net generation’ of students (Prensky, 2001; Oblinger & Oblinger, 2005; Tapscott & Williams, 2010). This article discusses the impact of digital technologies on each of Boyer’s dimensions of scholarship: discovery, integration, application and teaching. In each case the use of new technologies brings with it the possibility of new, more open ways of working, although this is not inevitable. The implications of the adoption of new technologies on scholarship are then discussed

Structural studies of MUC1 core related peptides

Polymorphic epithelial mucins are large complex glycoproteins which consist of a single polypeptide backbone, a large domain of which is usually made up of degenerate tandem repeats. One such molecule MUC1 is expressed at the surface of human mammary cells and is developmentally regulated and aberrantly expressed in tumours. These mucins have been identified as the target antigens for a number of murine monoclonal antibodies raised against a variety of immunogens including human milk products and breast tumour extracts. The anti-MUC1 antibodies have been shown to display tumour reactivity and have been used both as agents for imaging and in immunoassays to assess tumour burden and response to therapy. A number of these antibodies have been found to define epitopes within the tandem repeat of the protein core of the MUC1. Hydropathicity calculations and secondary structure predictions on the twenty amino acid tandem repeat of the MUC1 core protein have identified a hydrophilic domain Pro-Asp-Thr-Arg-Pro-Ala-Pro, which has a high probability of turn formation. It is within this domain that the epitopes of the anti-MUC1 antibodies are found. High field N.M.R. studies undertaken on an antigenic twenty amino acid peptide corresponding to the tandem repeat sequence of MUC1 in dimethyl sulphoxide have identified the presence of a type-I beta-turn in the region Pro-Asp-Thr-Arg. This turn overlaps the epitopes of all of the MUC1 antibodies characterised to date. In an attempt to identify more precisely the conformational requirements for binding to two anti-MUC1 antibodies, HMFG1 and HMFG2, the solution structures of several MUC1 core related peptides in dimethyl sulphoxide have been investigated. All of the peptides studied have been found to contain either beta-turns or modified turns which overlap their hydrophilic epitope domains. While these observations may provide some explanation for the observed reactivity of the different peptides, they give little insight into the precise structural requirements for antibody binding. Due to the flexibility of linear peptides in solution it is not possible to define the side chain conformations which are crucial to the processes of antibody recognition. In order to define the bound conformations of antigenic peptides using N.M.R. it is necessary to undertake experiments in the presence of antibody. Initial experiments have been performed in order to determine the conformation of the MUC 1 core related twenty amino acid peptide when bound to the anti-mucin antibody C595. In addition DNA coding for the variable domains of C595 has been cloned and sequenced in order to facilitate both expression of recombinant antibody binding fragments and the modelling of the binding site. These studies should provide a clearer understanding of the structural basis of antibody recognition of the peptides, and may give an insight into the specificity of anti-MUC1 antibodies for malignant cells

Accessible Inclusive Learning: Futures

The last chapter outlined some key approaches and challenges that we have seen when conducting research that seeks to make learning accessible to all. Here, we explore newer trends that are directing our current research and practice in this area. These promising directions include devising models for global networks, the potential to collect and use data to understand learning experiences in new ways, and new opportunities arising through artificial intelligence. By exploring current and recent projects around these areas, we also highlight some emerging tensions. Finally, we return to thinking about how we conduct research, considering how concepts of bricolage and guerrilla research can be an important part of our palette of approaches

Accessible Inclusive Learning: Foundations

One of the most persistent themes in discussions around technology in education is the idea that technology can affect access to learning. This can be seen as positive or negative, and it is often more complex than it seems. If computers can convert the text in a web page into spoken word, or the spoken words on a video into captions, have we made the learning accessible to deaf or blind students? Most likely we have made an important step in the right direction, but this might be only one challenge in the wider pedagogy and student experience. If MOOCs can teach thousands for free without any cost or entry requirements, does that mean they are increasing access? Perhaps, but are they also creating barriers for some through the pedagogical and technical design? In this chapter we will unpack how these issues have been tackled through research

Structural studies of MUC1 core related peptides

Polymorphic epithelial mucins are large complex glycoproteins which consist of a single polypeptide backbone, a large domain of which is usually made up of degenerate tandem repeats. One such molecule MUC1 is expressed at the surface of human mammary cells and is developmentally regulated and aberrantly expressed in tumours. These mucins have been identified as the target antigens for a number of murine monoclonal antibodies raised against a variety of immunogens including human milk products and breast tumour extracts. The anti-MUC1 antibodies have been shown to display tumour reactivity and have been used both as agents for imaging and in immunoassays to assess tumour burden and response to therapy. A number of these antibodies have been found to define epitopes within the tandem repeat of the protein core of the MUC1. Hydropathicity calculations and secondary structure predictions on the twenty amino acid tandem repeat of the MUC1 core protein have identified a hydrophilic domain Pro-Asp-Thr-Arg-Pro-Ala-Pro, which has a high probability of turn formation. It is within this domain that the epitopes of the anti-MUC1 antibodies are found. High field N.M.R. studies undertaken on an antigenic twenty amino acid peptide corresponding to the tandem repeat sequence of MUC1 in dimethyl sulphoxide have identified the presence of a type-I beta-turn in the region Pro-Asp-Thr-Arg. This turn overlaps the epitopes of all of the MUC1 antibodies characterised to date. In an attempt to identify more precisely the conformational requirements for binding to two anti-MUC1 antibodies, HMFG1 and HMFG2, the solution structures of several MUC1 core related peptides in dimethyl sulphoxide have been investigated. All of the peptides studied have been found to contain either beta-turns or modified turns which overlap their hydrophilic epitope domains. While these observations may provide some explanation for the observed reactivity of the different peptides, they give little insight into the precise structural requirements for antibody binding. Due to the flexibility of linear peptides in solution it is not possible to define the side chain conformations which are crucial to the processes of antibody recognition. In order to define the bound conformations of antigenic peptides using N.M.R. it is necessary to undertake experiments in the presence of antibody. Initial experiments have been performed in order to determine the conformation of the MUC 1 core related twenty amino acid peptide when bound to the anti-mucin antibody C595. In addition DNA coding for the variable domains of C595 has been cloned and sequenced in order to facilitate both expression of recombinant antibody binding fragments and the modelling of the binding site. These studies should provide a clearer understanding of the structural basis of antibody recognition of the peptides, and may give an insight into the specificity of anti-MUC1 antibodies for malignant cells

Inclusive volunteering: Exploring migrant participation in volunteerism

In partnership with Cork Volunteer Centre (CVC), this small-scale study explored migrants' experiences of volunteering in Cork city and county, including their motivations and pathways into volunteering, the benefits and challenges of volunteering, and the factors which facilitate or inhibit volunteering. The findings highlight the contribution which migrants make to local communities and the wide range of skills and experience which they bring to their volunteering roles. In line with international research, the study also indicates that volunteering can be an important aspect of two-way processes of integration

The Robertson v. Princeton Case: Too Important to Be Left to the Lawyers

Offers comments from eleven contributors on the Robertson family's donor rights suit against the Woodrow Wilson School of Public and International Affairs for violation of donor intent. Explores its effects on and implications for the nonprofit sector

Influence of positional correlations on the propagation of waves in a complex medium with polydisperse resonant scatterers

We present experimental results on a model system for studying wave propagation in a complex medium exhibiting low frequency resonances. These experiments enable us to investigate a fundamental question that is relevant for many materials, such as metamaterials, where low-frequency scattering resonances strongly influence the effective medium properties. This question concerns the effect of correlations in the positions of the scatterers on the coupling between their resonances, and hence on wave transport through the medium. To examine this question experimentally, we measure the effective medium wave number of acoustic waves in a sample made of bubbles embedded in an elastic matrix over a frequency range that includes the resonance frequency of the bubbles. The effective medium is highly dispersive, showing peaks in the attenuation and the phase velocity as functions of the frequency, which cannot be accurately described using the Independent Scattering Approximation (ISA). This discrepancy may be explained by the effects of the positional correlations of the scatterers, which we show to be dependent on the size of the scatterers. We propose a self-consistent approach for taking this "polydisperse correlation" into account and show that our model better describes the experimental results than the ISA