The study of a prokaryotic glycolytic enzyme

The overall objective of this project is to generate novel carbohydrate binding proteins for use in glycoprotein analysis which are amenable to large scale production. The approach used here is the modification of prokaryotic glycolytic enzymes. Their enzymatic activity will be eliminated while hoping they still retain their binding capabilities. These proteins will be immobilized onto different surfaces to generate advanced bioanalytical platforms which will have huge commercial potential in the field of glycoanalysis

Glycolytic enzymes - novel carbohydrate binding proteins for glycoprotein analysis

•The cloning, expression, purification and characterisation of recombinant prokaryotic glycolytic enzymes •The mutagenesis of prokaryotic glycolytic enzymes to generate novel recombinant carbohydrate binding proteins •The characterisation of the binding profile of the novel recombinant carbohydrate binding protein

Defining Mother-Infant Synchrony in a Speech and Song Context

The objective of this study was to examine the behaviours observed within mother-infant dyads during speech and song play. Previous research has suggested that caregivers convey emotional meaning through vocalizations and emanate behaviours that synchronize interactions with their infants (Dissanayake, 2000; Reyna & Pickler, 2009). Research has also suggested that infants prefer infant-directed singing over speaking and that song can be used to regulate infants’ states of arousal (Nakata & Trehub, 2004). The current study was designed to extend the literature on mother-infant interactions by having mothers play with their infants while singing or speaking to them. The speech context was elicited by asking mothers to play with their infants as they normally would, while the song context was elicited by asking them to sing nursery rhymes of their choice while they played. Subsequently, a researcher entered the room to distract the mother by asking a series of questions. Both maternal and infant behaviour during speech and song were coded for frequency of occurrence; infant behaviour was further coded during the disruption. Results showed that maternal behaviour, including infant referencing, object referencing, and pausing, occurred more frequently in response to speech than to song. Infant behaviour, including positive affect, attention, and turn taking, showed no differences across the two contexts. Additionally, results showed no difference in infant behaviour during the disruption based on the prior context. The findings from the current study give insight into the types of behaviours mothers pair with vocalizations as well as the function of these behaviours

Strain-induced structural instability in FeRh

We perform density functional calculations to investigate the structure of the inter-metallic alloy FeRh under epitaxial strain. Bulk FeRh exhibits a metamagnetic transition from a low-temperature antiferromagnetic (AFM) phase to a ferromagnetic (FM) phase at 350K, and its strain dependence is of interest for tuning the transition temperature to the room-temperature operating conditions of typical memory devices. We find an unusually strong dependence of the structural energetics on the choice of exchange-correlation functional, with the usual local density approximation (LDA) yielding the wrong ground-state structure, and generalized gradient (GGA) extensions being in better agreement with the bulk experimental structure. Using the GGA we show the existence of a metastable face-centered-cubic (fcc)-like AFM structure that is reached from the ground state body-centered-cubic (bcc) AFM structure by following the epitaxial Bain path. We predict that this metastable fcc-like structure has a significantly higher conductivity than the bcc AFM phase. We show that the behavior is well described using non-linear elasticity theory, which captures the softening and eventual sign change of the orthorhombic shear modulus under compressive strain, consistent with this structural instability. Finally, we predict the existence of an additional unit-cell-doubling lattice instability, which should be observable at low temperature.Comment: 10 pages, 7 figure

Production of lectin-affinity matrices for process-scale glycoprotein purification

A selection of prokaryotic lectins with a variety of glycan specificities and affinities have been identified, cloned, expressed in Eschericia coli and characterised. The aims of this project are to: - express the lectins at 1L scale to produce sufficient quantities for immobilisation studies (~100 mg) - immobilisethelectinsonSepharose - evaluate lectin performance on column by monitoring their ability toreproducibly capture and elute glycoprotein glycoforms

Lectin based glycoprotein analysis

Many of the biopharmaceutical therapeutics entering the market and currently in clinical trails are recombinant glycoprotein molecules, the glycan moieties of which have a significant impact on efficacy and immunogenicity. The cell culture techniques required to produce these glycoproteins often result in products that are heterogeneous with respect to glycan content. This inconsistency ultimately leads to increased production costs and restricts patient accessibility to these therapeutics. To overcome these difficulties novel analytical platforms facilitating rapid in-process monitoring and product quality control are essential. Work undertaken within the Centre for Bioanalytical Sciences (CBAS) seeks to exploit the microbial world as a source of novel biorecognition elements to produce such platforms

Exploiting prokaryotic chitin-binding proteins for glycan recognition

• The cloning, expression and characterisation of prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus luminescens Microfluidics and Photorhabdus asymbiotica • Development of an assay to assess the activity of chitin-binding proteins • Mutagenesis of chitin-binding proteins to alter glycan recognition pattern

Genetically enhanced recombinant lectins for glyco-selective analysis and purification

- Generation of a library of recombinant prokaryotic lectins (RPL’s) through random mutagenesis of the carbohydrate binding sites of bacterial lectins. - Characterisation of mutant lectins with respect to structure and specificity - Provision of mutant RPL’s with enhanced affinity and/or altered specificity, alongside wild-type RPL’s, for glycoprotein analysis and purificatio

Gold nano-particle modified silica monolithic micro-columns for selected chromatographic and biological applications.

Monolithic microcolumns and especially silica monoliths are showing several advantages compared to classical particle packed and organic polymeric monolithic columns: ease of production and functionalisation, excellent mechanical and thermal stability. Morphology of the monolithic columns can easily be tuned by simply changing the compositions of reaction mixtures. High porosity and interconnected flow-through pores ensure low back pressures at higher flow rates so increasing reaction speeds. High salt resistance allows use water based buffer solutions without any swelling of the stationary phase, large biomolecules can be utilised and conditions to prevent denaturation and comformation changes of these biomolecules can be maintained. Introduction of gold nano-particles on the surfaces of silica monoliths allows increase of the surface areas and alows creation of new, exotic surfaces. Gold shows strong affinity towards thiol groups, which can be found in different biomolecules so utilisation of this phenomena would allow production of micro-reactors and bioreactors in order to mimic biological reactions happening in living organisms and large biological systems. Silica monoliths were synthesised using classical sol-gel process. In order to immobilise gold nano-particles, surfaces of the silica monoliths were amminated using standard silanisation reaction with 3-aminopropyl-methyl-diethoxysilane. 20 nm citrate stabilised gold nano-particles were immobilised on the surfaces afterwards. Depending on the desired application, gold nano-oparticle modified silica monoliths were functionalised afterwards. Immobilisation of ionic species such as amino acids and small peptides would allow creation of stationary phase for ion chromatography, retention of enzymes and other biologically active molecules would allow to create micro-reactors. Leaving gold nano-particles unmodified would make ideal stationary phase for micro-extraction. These modified monoliths were characterised using microscopy techniques, such as scanning electron microscopy (SEM) and field emission SEM. They were used to characterise morphology of the monoliths as well as to evaluate the coverage of the surface with gold nano-particles. The fabricated stationary phases were used for selected biological and chromatographic applications (incorporanting classical chromatographic techniques in order to evaluate the performance of these new modified monolithic materials)

Regions of the Cry1Ac toxin predicted to be under positive selection are shown to be the carbohydrate binding sites and can be altered in their glycoprotein target specificity

The cry gene family, is a large family of homologous genes from Bacillus thuringiensis. Studies have examined the structural and functional relationships of the Cry proteins. They have revealed several residues in domains II and III that are important for target recognition and receptor attachment. In 2007 Wu, Jin-Yu et al employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins. They identified positively selected residues, which are all located in Domain II or III. Figure 1 shows a protein sequence alignment between domain II and III of Cry1Ac and Cry1Aa. This highlights the areas which are thought to be under positive selection. Cry1Ac and Cry1Aa are structurally very similar and they both bind to a variety of N-aminopeptidases (APN’s) in different insect species. However Cry1Aa has a higher specificity for the cadherin like receptor HevCalP and Cry1Ac binds to N-acetylgalactosamine (GalNAc) on the surface of APN’s. Differences in the binding of the two toxins has been shown in an in-direct toxin-binding assay where GalNAc completely abolished toxin binding of Cry1Ac but had no effect on the binding of Cry1Aa. The binding site has been shown to be located in the third domain of Cry1Ac. Some of these sites correlate with the positively selected residues found by Wu et al 2007 in Cry1Aa. Our aim was to use the comparison of the toxins to analyse the potential to alter the binding specificity of Cry1Ac and its domains. In this work we identified critical amino acid residues for this objective