Applying Online: Technological Innovation for Income Support Programs in Four States

A study examining the development, implementation, and best practices for online applications for public benefits programs in California, Georgia, Pennsylvania, and Washington based on interviews with state agencies and community-based organizations

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

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

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

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

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

Recombinant prokaryotic lectins : enhanced tools for glycoprotein analysis and purification

More than 50% of proteins are glycosylated and glycans are known to mediate a wide range of biological processes. With this knowledge, there has been an explosion of interest in the field of glycoproteomics. There is a need for tools that enable efficient isolation of glycoproteins from biological samples, where they are usually only present at low levels, to enable their identification and analysis. Changes in glycosylation ptterns of biomolecules and cells are also associated with many diseases such as cancer and rheumatoid arthritis. Tools capable of sensitive detection of such changes would have significant potential in the field of diagnosics. In addition, many biopharmaceuticals are glycosylated and the glycosylation impacts their clinical properties. The industry needs tools for sensitive product analysis and selective purification of op/mally glycosylated product to meet regulatory requirements and to bring safer, more effective ,products to patents. Recombinant Prokaryotic Lectins (RPL’s) offer new opportunities to develop enhanced glycoselective tools for glycoprotein analysis and purification and to overcome the limitations that have restricted the applications of plant lectins

Drugs associated with cataract formation represent an unmet need in cataract research

Decreased light transmittance through the ocular lens, termed cataract, is a leading cause of low vision and blindness worldwide. Cataract causes significantly decreased quality of life, particularly in the elderly. Environmental risk factors, including aging, UV exposure, diabetes, smoking and some prescription drugs, are all contributors to cataract formation. In particular, drug-induced cataract represents a poorly-addressed source of cataract. To better understand the potential impact of prescription drugs on cataract, we analyzed publicly-available drug prescriptions data from the Australian Pharmaceutical Benefits Scheme. The data was analyzed for the 5-year period from July 2014 to June 2019. Analyses included the number of prescriptions for each drug, as well as the associated government and total prescription costs. The drugs chosen for analysis belonged to any of four broad categories—those with known, probable, possible or uncertain association with cataract in patients. The analyses revealed high prescription rates and costs for drugs in the Known category (e.g., steroids) and Possible category (e.g., psychotropic drugs). Collectively, these data provide valuable insights into specific prescription drugs that likely contribute to the increasing annual burden of new cataract cases. These data highlight the need—as well as new, stem cell-based opportunities—to elucidate molecular mechanisms of drug-induced cataract formation