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

Desensitization to medical examining equipment through learner control in a child with severe mental retardation

While the psychological literature is replete with examples of desensitization to phobic stimuli, few studies have documented successful interventions conducted with individuals identified as mentally retarded. The study describes desensitization to basic medical examining equipment in a youngster with severe mental retardation and autistic tendencies. Following desensitization to feared stimuli through the repeated exposure of baseline, intervention was initiated on the remaining feared stimuli through a learner control technique which combined modeling, behavioral rehearsal, and a variation of contact desensitization (Ritter, 1968). Results of a return-to-baseline design suggested that the medical examinations feared most by the child were of the same functional response class and that they did respond positively to intervention. Discussion of the methods and results provides practical implications for health professionals as well as offering hypotheses regarding the potential communicative and adaptive functions phobic manifestations serve in persons who experience significant handicapping conditions

Benefits and barriers of cancer practitioners discussing physical activity with their cancer patients

Our aim was to synthesise the existing empirical literature and theoretical perspectives on the physical activity (PA) promotion practices and determinants of cancer clinicians and health professionals. We conducted a narrative review of theory and evidence to develop practice recommendations for improving the promotion of PA to cancer patients. Surveyed health professionals were aware of many benefits of PA for their cancer patients, although only ~40 % promoted PA to selected cancer patients. Walking was the most commonly promoted form of PA, with this promoted to assist patients control their weight and cardiovascular health risk. Barriers to promotion of PA included lack of time and knowledge of PA and behaviour change skills. Health professionals appear interested in promoting PA to their cancer patients, yet encounter several barriers. Further research is warranted to assist health professionals improve their PA promotion. An adapted reflective-impulsive model of social behaviour shows promise for assisting health professionals overcome barriers and provides an evidence-based theoretical framework for improving communication with patients. Universities, hospitals and/or health-care accreditation organisations also have important roles to play in assisting health professionals improve their promotion of PA to patients

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

The investigation of a recombinant GalNAc binding protein from bacillus thuringiensis as a tool for glycan analysis and detection

Changes in the structures of glycans on the surfaces of eukaryotic cells can be important biomarkers for developmental or disease states. Improved methods are needed for the detection and analysis of alterations in glycan structures. Carbohydrate binding proteins such as lectins have potential for the recognition of changes in glycan structure. Host-pathogen interactions frequently involve the recognition of host carbohydrates by proteins of bacteria or viruses. Many bacterial toxins have evolved to interact with host cell receptors or with a specific tissue due to lectin like properties. The toxins from Bacillus thuringiensis have been shown to have carbohydrate binding abilities, in particular N-Acetylgalactosamine (GalNAc) has been shown to inhibit the binding of the toxin Cry1Ac. GalNAc has been shown to be an important marker in many diseases such as breast cancer and colon carcinogenesis. Moreover, changes in GalNAc glycosylation have been identified in many disorders such as cystic fibrosis, neuromuscular disorders and nephropathy. Here we describe the purification of a GalNAc binding protein of bacterial origin that may have potential in the development of diagnostic assays

Exploitation of siderophores for the speciation of iron

Iron is essential for life. It acts as an electron donor/acceptor in metabolic processes facilitated by its variable valency. Although vital, it is toxic at high levels due to Fe2+ oxidation. Iron toxicity is a concern as it can affect growth and product yields in animal cell culture. Siderophores are high affinity Fe3+ chelators produced by microorganisms. This affinity gives them the potential to be used as a basis in platforms to detect and speciate iron in industrial cell culture. Rhizobactin 1021 is of interest due to its decanoic acid “tail” that is not involved in chelation which makes it an ideal target for immobilisation

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

Transcriptome analyses reveal reduced hepatic lipid synthesis and accumulation in more feed efficient beef cattle

peer-reviewedThe genetic mechanisms controlling residual feed intake (RFI) in beef cattle are still largely unknown. Here we performed whole transcriptome analyses to identify differentially expressed (DE) genes and their functional roles in liver tissues between six extreme high and six extreme low RFI steers from three beef breed populations including Angus, Charolais, and Kinsella Composite (KC). On average, the next generation sequencing yielded 34 million single-end reads per sample, of which 87% were uniquely mapped to the bovine reference genome. At false discovery rate (FDR)  2, 72, 41, and 175 DE genes were identified in Angus, Charolais, and KC, respectively. Most of the DE genes were breed-specific, while five genes including TP53INP1, LURAP1L, SCD, LPIN1, and ENSBTAG00000047029 were common across the three breeds, with TP53INP1, LURAP1L, SCD, and LPIN1 being downregulated in low RFI steers of all three breeds. The DE genes are mainly involved in lipid, amino acid and carbohydrate metabolism, energy production, molecular transport, small molecule biochemistry, cellular development, and cell death and survival. Furthermore, our differential gene expression results suggest reduced hepatic lipid synthesis and accumulation processes in more feed efficient beef cattle of all three studied breeds