The use of chitin binding proteins for glycoprotein analysis

The focus of the pharmaceutical industry has dramatically shifted in the past number of years. Traditional drugs were synthesised using chemical reactions have been replaced by recombinant glycoprotein molecules. These potential recombinant glycoprotein therapeutics display oligosaccharide structures on their surfaces that are recognised by their target host. The specific glycan moieties on the surface of the molecules vary dramatically and have a large impact on the efficacy of the drug. The development of bioanalytical tools to identify and separate the species of glyco-forms present in a preparation of the glycoprotein therapeutic will significantly help to advance the quality and effectiveness of recombinant glycoprotein molecules. Traditionally lectins, isolated from plants, had been used to profile sugar species displayed on glycoproteins. I have explored the use of prokaryotic chitin binding proteins (CBPs) to investigate structures on glycoproteins

Design of new highly functional polymer grafted polyhipes for proteins immobilization

PolyHIPE have proven to be useful in a large variety of applications included column filtration/separation, supported organic chemistry, as media for tissue engineering and 3D cell culture.1 The ability to conveniently modify pHIPE surfaces with functional groups is essential to opening new applications areas. The most promising method to conveniently modify pHIPE surface with a high density of functional groups is the “grafting from” approach. Stable polymer brushes covalently attached to the surface posses excellent mechanical and chemical robustness and offer the flexibility to introduce a large variety of functional monomers.2 We developed a new and unique pHIPE platform by incorporation of a polymerizable monomer with amino group into the HIPE available for different post in situ polymerization. The pHIPE with amino groups on the surface (pHIPE-NH2) can be directly used for the ring opening polymerization of amino acids N-carboxyanhydrates (NCAs) monomers to make pHIPE-g-polypeptide (such as pHIPE-g-poly(L-Benzyl Glutamate)) or easily converted to an atom transfer radical polymerization (ATRP) initiator for activators generated electron transfer (AGET) ATRP of tert-Butyl acrylate monomers. The polymers grafted can be deprotected to form pHIPE-g-poly(glutamic acid) or pHIPE-g-poly(acrylic acid) with reactive groups, on the surface of the pHIPE, available for further bioconjugation

Development of new bioaffinity phases for glycoprotein separation and analysis

The past decade has seen an appreciation of the critical biological significance of glycosylation and the impact that glycans can have on the efficacy, stability and immunogenicity of important glycoproteins. For the biopharma industry the level of product characterization and process monitoring demanded, particularly in the context of glycosylation, is set to increase and will become an even more significant issue with the entry of biosimilar products into the biopharmaceutical arena. In order to meet these demands of the regulatory biopharmaceutical and generic producers alike require rapid , sensitive and high throughput techniques to enable detailed glycoprotein separation and analysis. It is extremely difficult to separate closely related glycoforms using standard chromatographic methods, such as ion exchange chromatography (IEX), gel exclusion chromatography (GEC) and hydrophobic interaction chromatography (HIC). Here , we report the development of a series of new bio-affinity based phases that are capable of separating closely related glycoproteins/glycoproteins. These phases get their selectivity from a number of recombinant prokaryotic bioligands called lectins. Lectins are proteins that are capable of recognizing and binding reversibly to specific glycan structures. While lectin binding affinities for monosaccharides are generally low they bind to disaccharides and more complex oligosaccharide structures with significantly higher affinities and exquisite specificity

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

Protein immobilization on highly functional polymer grafted polyHIPEs

PolyHIPE have proven to be useful in a large variety of applications included column filtration/separation, supported organic chemistry, as media for tissue engineering and 3D cell culture.[1] We developed a new pHIPE platform by incorporation of a polymerizable monomer with amino group into the HIPE available for different post polymerizations.[2] The pHIPE with amino groups on the surface (pHIPE-NH2) can be directly used for the ring opening polymerization of amino acids N-carboxyanhydrates (NCAs) to obtain pHIPE-g- polypeptides or easily converted to an atom transfer radical polymerization (ATRP) initiator for activators generated electron transfer (AGET) ATRP of tert-butyl acrylate. The polymers grafted were deprotected to form or pHIPE-g-poly(acrylic acid) with reactive groups on the surface of the pHIPE, available for bioconjugation of fluorescent proteins such as enhanced green fluorescent protein (eGFP

Risks and Outcomes Associated with Disorganized/Controlling Patterns of Attachment at Age Three in the NICHD Study of Early Child Care and Youth Development

Disorganized/controlling attachment in preschool has been found to be associated with maternal and child maladjustment, making it of keen interest in the study of psychopathology. Additional work is needed, however, to better understand disorganized/controlling attachment occurring as early as age three. The primary aims of this study were to evaluate risk factors and outcomes associated with disorganized/controlling behavior at age three and to evaluate the risk factors and outcomes differentiating the four subtypes of disorganized/controlling attachment. Analyses were conducted with the first two phases of the NICHD Study of Early Child Care and Youth Development, a prospective study of 1,364 children from birth. At 36 months of age, across the attachment-relevant domains of maternal well-being, mother-child interactions, and child social adaptation, the disorganized/controlling group evidenced the most maladaptive patterns in comparison to both secure and insecure-organized groups. At 54 months of age, the disorganized/controlling group displayed the highest levels of internalizing and externalizing behavior problems, as rated by mothers and teachers, and the lowest quality relationships with teachers. Significant differences found among the disorganized/controlling subtypes indicated that the behaviorally disorganized and controlling-punitive subtypes had more maladaptive patterns across variables than did the controlling-caregiving and controlling-mixed subtypes

Cesagen response to Nuffield Council on bioethics consultation on novel neurotechnologies:intervening in the brain

In what follows, we do not answer every question [by the Nuffield Council on Bioethics). We first proceed with our comments, referring to the numbered questions as appropriate. Thereafter, we give a case study from recent studies within Cesagen to illustrate more general insights for public policy. Case study 1 illustrates some of the complications that arise in public consultation about human enhancement, in particular, with reference to idealistic perceptions which are strongly influenced by long-term popular imaginations about the future of humans and their societies. As we said in a response to a previous consultation, our position is that attention needs to be paid to how the technologies and the associated issues are framed – ethically, politically, scientifically, and by whom. This includes how a given technology is itself described (typically well before it actually exists, if it comes to do so); the claims made for its purported benefits; how stakeholders are conceptualised; how social-cultural aspects will evolve. Such framing is not exclusively a scientific and technological matter but involves cultural and social imaginations as well as artistic ones

Indiscriminate behavior observed in the strange situation among institutionalized toddlers : relations to caregiver report and to early family risk

Socially disinhibited or indiscriminate behavior (IB) has traditionally been investigated using caregiver reports. More recently, an observational measure based on the Strange Situation Procedure, the Rating of Infant and Stranger Engagement (RISE), was validated in home-reared at-risk children. The present study aimed to validate the RISE in an institutionally reared sample using the caregiver report, to assess whether IB assessed with the RISE was elevated among the institutionalized children, and to explore potential risk factors associated with IB. The study was conducted among 74 institutionalized toddlers, aged 11 to 30 months. Sociodemographic questionnaires were used to assess pre-admission experiences, and aspects of institutional placement were coded from the children’s files in the institution and staff’s report. Institutionalized children displayed high frequencies of IB as assessed on the RISE, and this instrument was validated against caregiver report. Pre-admission experiences of the institutionalized children in their biological families, namely prenatal risk and maternal emotional-neglect risk, predicted IB. Results suggest that the RISE is adequate to use among institutionally reared toddlers and point to aspects of the early familial environment that may be implicated in IB

Staying just one step ahead: providing care for patients with motor neurone disease

Introduction: There is limited information about the experiences and educational needs of health professionals who may be required to provide care for people with Motor Neurone Disease(MND) especially in the later stages of the disease. The aim of this study was to determine the experiences of, and need for, education of these health professionals. Methods: Interviews and focus groups were conducted with 31 health professionals with some experience in providing palliative care for people with MND. Thematic content analysis was used to identify common themes. Results: A key theme, Just One Step Ahead, emerged, that describes the central capability health professionals identified as necessary to help individuals plan and prepare for disease and lifestyle changes just before they arise. Two subthemes also emerged: Expertise in MND and Bespoke Communication. Expertise in MND described the required understanding of the disease and the particular individual’s version of the disease to allow the health professional to plan, advise, support and anticipate the needs of the person living with MND. Bespoke Communication was the facility to tailor care messages sensitively and effectively to the range of people involved in care (patients, family, healthcare team members).Conclusions: Care of people with MND requires up-to-date expertise about the disease and skilled communication abilities to manage complexity and change. Timely and focused education and specialist MND support for care providers are essential to tailored and responsive care and a widely available education programme has been developed to address these needs