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Wetherby, United Kingdom

Kawakami M.,Japan Advanced Institute of Science and Technology | Kawakami M.,PRESTO of Japan Science and Technology Corporation | Taniguchi Y.,Japan Advanced Institute of Science and Technology | Taniguchi Y.,Japan Society for the Promotion of Science | And 4 more authors.
Langmuir | Year: 2010

In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the "squeezing effect", becomes significant, and. the resonance peak, of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved. © 2009 American Chemical Society.

Stadler L.K.J.,Section of Experimental Therapeutics | Stadler L.K.J.,Medical Research Council Laboratory of Molecular Biology | Tomlinson D.C.,St Jamess Hospital | Tomlinson D.C.,University of Leeds | And 5 more authors.
Cell Death and Disease | Year: 2014

The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-Associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-Apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold-Stefin A quadruple mutant-to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-Associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-Apoptotic effector protein observed in this study.

Sharma A.,University of Leeds | Leach R.N.,University of Leeds | Leach R.N.,Covance | Gell C.,University of Leeds | And 11 more authors.
Nucleic Acids Research | Year: 2014

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ70 and σ54, the domain movements of the latter have evolved to require an activator ATPase. © 2014 © The Author(s) 2014. Published by Oxford University Press.

Agency: GTR | Branch: Innovate UK | Program: | Phase: Department of Trade & Industry | Award Amount: 209.22K | Year: 2006

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Rapid generation and validation of three distinct and highly specific Affimer binders to Zika virus within just thirteen weeks of receiving the virus target.The Affimer reagents bind a protein called NS1 which is diagnostic of the early, acute stage of Zika infection and for which no validated antibody is available ...

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