Monitoring complex formation by relaxation-induced pulse electron paramagnetic resonance distance measurements

Funding: EPSRC DTC and Wellcome (099149/Z/12/Z).Biomolecular complexes are often multimers fueling the demand for methods that allow unraveling their composition and geometric arrangement. Pulse electron paramagnetic resonance (EPR) spectroscopy is increasingly applied for retrieving geometric information on the nanometer scale. The emerging RIDME (relaxation-induced dipolar modulation enhancement) technique offers improved sensitivity in distance experiments involving metal centers (e.g. on metalloproteins or proteins labelled with metal ions). Here, a mixture of a spin labelled ligand with increasing amounts of paramagnetic CuII ions allowed accurate quantification of ligand-metal binding in the model complex formed. The distance measurement was highly accurate and critical aspects for identifying multimerization could be identified. The potential to quantify binding in addition to the high-precision distance measurement will further increase the scope of EPR applications.Publisher PDFPeer reviewe

Binding dynamics of a monomeric SSB protein to DNA : a single-molecule multi-process approach

People Programme of the European Union’s Seventh Framework Programme [REA 334496 to B.E.B.]; Leonardo da Vinci European Union Programme (to M.F.G.); Wellcome Trust [099149/Z/12/Z, 091825/Z/10/Z]. Funding for open access charge: Wellcome Trust; University of St Andrews.Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between highdensity binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.Publisher PDFPeer reviewe

Autonomous Sensory Meridian Response (ASMR) as a marketing tool : an examination of the online phenomenon' s potential in the promotion mix of slow tourism destinations

Autonomous Sensory Meridian Response (ASMR) is an online phenomenon that is becoming increasingly popular and has been brought to the attention of the public by brands using it to advertise their products. The goal of this thesis is to evaluate the potential of this phenomenon for promoting slow tourism destinations and to develop recommendations for action based on the findings of the study. The research question the study aims to answer is how slow tourism destinations can use ASMR as a marketing tool in their promotion mix. To answer this question, a literature review was carried out and an online survey addressed to the ASMR community was conducted. The study found that in order to use ASMR as a marketing tool, slow tourism destinations need to show an appreciation for ASMR by producing content that is valuable to the community and appeals to the widest possible audience. This can be achieved by acknowledging and using the preferences of the community as determined in this study, making appropriate themes, such as nature, wellness or slowness, the focus of the marketing content as well as choosing the kind of ASMR marketing most suitable to the destination. While ASMR can be used for offline communication, such as on TV or radio, the study found that the ASMR community greatly preferred online communication, either through sponsorship or, to a lesser extent, though self-produced content

Pulse dipolar electron paramagnetic resonance spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

Funding: Leverhulme Trust - RPG-2018397; Biotechnology and Biological Sciences Research Council - BB/M010996/1; Engineering and Physical Sciences Research Council - EP/N509759/1; Wellcome Trust - 204821/Z/16/Z.Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.Publisher PDFPeer reviewe

The Importance of Being Profiled: Improving Drug Candidate Safety and Efficacy Using Ion Channel Profiling

Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008)

Association mapping in Scandinavian winter wheat for yield, plant height and traits important for second-generation bioethanol production

A collection of 100 wheat varieties representing more than 100 years of wheat-breeding history in Scandinavia was established in order to identify marker-trait associations for plant height, grain yield and biomass potential for bioethanol production. The field-grown material showed variations in plant height from 54 to 122 cm and in grain yield from 2 to 6.61 t ha-1. The release of monomeric sugars was determined by high-throughput enzymatic treatment of ligno-cellulosic material and varied between 0.169 and 0.312 g/g dm for glucose and 0.146 and 0.283 g/g dm for xylose. As expected, plant height and grain yield showed to be highly influenced by genetic factors with repeatability (R) equal to 0.75 and 0.53 respectively, while this was reduced for glucose and xylose (R=0.09 for both) . The study of trait correlations showed how old, low-yielding, tall varieties released higher amounts of monomeric sugars after straw enzymatic hydrolysis, showing reduced recalcitrance to bioconversion compared to modern varieties. 93 lines from the collection were genotyped with the DArTseq® genotypic platform and 5525 markers were used for genome-wide association mapping. Six QTLs for grain yield, plant height and glucose released from straw were mapped. One QTL for plant height was previously reported, while the remaining QTLs constituted new genomic regions linked to trait variation. This paper is one of the first studies in wheat to identify QTLs that are important for bioethanol production based on a genome-wide association approach

project report Promise2007

Das Projekt Promise2007 befasste sich mit der Erstellung und Auswertung einer Statistik zur Mitgliedersituation im Berufsverband Medizinischer Informatiker e.V.. Mit dem Ziel mehr über die Mitglieder und ihre derzeitige Situation zu erfahren wurde das Projekt an der Fachhochschule Hannover initiiert. Statistisch erfasst wurden Fragen zum Beschäftigungsverhältnis, zu Aus- und Weiterbildung, der beruflichen Situation und persönliche Angaben. Die Ergebnisse wurden ausgewertet und daraus wichtige Erkenntnisse für den BVMI e.V. abgeleitet, welche auf die weitere Verbandsarbeit Einfluss nehmen

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. © 2013 Neal et al