Whispering Gallery Modes in Standard Optical Fibres for Fibre Profiling Measurements and Sensing of Unlabelled Chemical Species

Whispering gallery mode resonances in liquid droplets and microspheres have attracted considerable attention due to their potential uses in a range of sensing and technological applications. We describe a whispering gallery mode sensor in which standard optical fibre is used as the whispering gallery mode resonator. The sensor is characterised in terms of the response of the whispering gallery mode spectrum to changes in resonator size, refractive index of the surrounding medium, and temperature, and its measurement capabilities are demonstrated through application to high-precision fibre geometry profiling and the detection of unlabelled biochemical species. The prototype sensor is capable of detecting unlabelled biomolecular species in attomole quantities

Evolution of PHDs as oxygen sensors: mechanistic and structural studies of the PHD of Trichoplax adhaerens, the simplest animal, and mechanistic studies of a PHD-like enzyme of the protist Monosiga brevicollis

This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The α/β-heterodimer HIF (Hypoxia Inducible Factor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF α-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 Å), one containing a Trichoplax adhaerens HIFα subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C4-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIFα hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.</p

Whispering Gallery Modes in Standard Optical Fibres for Fibre Profiling Measurements and Sensing of Unlabelled Chemical Species

Whispering gallery mode resonances in liquid droplets and microspheres have attracted considerable attention due to their potential uses in a range of sensing and technological applications. We describe a whispering gallery mode sensor in which standard optical fibre is used as the whispering gallery mode resonator. The sensor is characterised in terms of the response of the whispering gallery mode spectrum to changes in resonator size, refractive index of the surrounding medium, and temperature, and its measurement capabilities are demonstrated through application to high-precision fibre geometry profiling and the detection of unlabelled biochemical species. The prototype sensor is capable of detecting unlabelled biomolecular species in attomole quantities

Evolution of PHDs as oxygen sensors: mechanistic and structural studies of the PHD of Trichoplax adhaerens, the simplest animal, and mechanistic studies of a PHD-like enzyme of the protist Monosiga brevicollis

This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The &alpha;/&beta;-heterodimer HIF (Hypoxia Inducible Factor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF &alpha;-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 &Aring;), one containing a Trichoplax adhaerens HIF&alpha; subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C4-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIF&alpha; hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.This thesis is not currently available in OR

The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens

The hypoxic response in humans is mediated by the hypoxia-inducible transcription factor (HIF), for which prolyl hydroxylases (PHDs) act as oxygen-sensing components. The evolutionary origins of the HIF system have been previously unclear. We demonstrate a functional HIF system in the simplest animal, Trichoplax adhaerens: HIF targets in T. adhaerens include glycolytic and metabolic enzymes, suggesting a role for HIF in the adaptation of basal multicellular animals to fluctuating oxygen levels. Characterization of the T. adhaerens PHDs and cross-species complementation assays reveal a conserved oxygen-sensing mechanism. Cross-genomic analyses rationalize the relative importance of HIF system components, and imply that the HIF system is likely to be present in all animals, but is unique to this kingdom

Droplet-based microfluidic synthesis of anisotropic metal nanocrystals

10.1002/smll.200901453Small5242828-283