The Relationship Between Agency Characteristics and Quality of Home Care

ABSTRACT. Background. This project assessed the relationship between home care quality indicators HCQIs) and agency characteristics. Methods. Twelve agencies completed a mailed survey on a variety of characteristics, including size of their caseload and for-profit (FP) status of contracted service providers. The HCQIs were derived from standardized assessments completed voluntarily for home care clients in Ontario and in Manitoba, Canada. Results. The average caseload was 121.3 clients per case manager, and over 40% of nursing, personal support and therapy providers were considered FP. For individual HCQIs, few correlations were statistically significant. An overall summary measure of quality was correlated with the size of the population served (r = _0.80; p \u3c 0.05) and the number of clients per case manager (r = _0.56; p \u3c 0.1). Conclusion. These data represent unique information on home care quality and organizational characteristics in Canada. The question remains as to how best to use HCQI data to inform practice in an era of limited resources and increasing caseloads

Functional and computational identification of a rescue mutation near the active site of an mRNA methyltransferase

RNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifcations at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and sufer from low turnovers. We have investigated the efects of homologous and orthologous active-site mutations on both stability and transferase activity, and identifed new functional motifs in the interaction network surrounding the catalytic lysine. Our fndings suggest that despite their low catalytic efciency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defned third shell around the active site have strong efects on folding, stability and activity in the variant enzymes, mostly via network-mediated efects

COPASAAR – A database for proteomic analysis of single amino acid repeats

BACKGROUND: Single amino acid repeats make up a significant proportion in all of the proteomes that have currently been determined. They have been shown to be functionally and medically significant, and are associated with cancers and neuro-degenerative diseases such as Huntington's Chorea, where a poly-glutamine repeat is responsible for causing the disease. The COPASAAR database is a new tool to facilitate the rapid analysis of single amino acid repeats at a proteome level. The database aims to simplify the comparison of repeat distributions between proteomes in order to provide a better understanding of their function and evolution. RESULTS: A comparative analysis of all proteomes in the database (currently 244) shows that single amino acid repeats account for about 12–14% of the proteome of any given species. They are more common in eukaryotes (14%) than in either archaea or bacteria (both 13%). Individual analyses of proteomes show that long single amino acid repeats (6+ residues) are much more common in the Eukaryotes and that longer repeats are usually made up of hydrophilic amino acids such as glutamine, glutamic acid, asparagine, aspartic acid and serine. CONCLUSION: COPASAAR is a useful tool for comparative proteomics that provides rapid access to amino acid repeat data that can be readily data-mined. The COPASAAR database can be queried at the kingdom, proteome or individual protein level. As the amount of available proteome data increases this will be increasingly important in order to automate proteome comparison. The insights gained from these studies will give a better insight into the evolution of protein sequence and function

Space for a Change? An Exploration of Power, Privilege and Transformative Pedagogy in a Gap Year Education Programme in South America

This thesis is situated in the context of a gap-year education programme operated in Bolivia and Peru by a US-based organisation. Inspired by Paolo Freire’s social-emancipatory educational ethos, the organisation transposes his Pedagogy of the Oppressed (1970) from literacy programmes for Brazilian peasants to a very different context: into attempts to transform privileged 18 to 21-year-olds from the “developed” world into critical global citizens, ready to challenge social injustices after three months in “developing” countries. This Freirean sentiment is unusual in the gap year industry, as well as in the academic literature on transformative learning which has emphasised personal transformation, overlooking social change and power relations. My thesis addresses this oversight, engaging with the concept of power as an ‘invisible’, symbolic network of social boundaries that defines ‘fields of possibility’ (Hayward, 1998), shaping what happens during the programme. Much research into transformative educational experiences focuses on the learning outcomes of self-identified transformed learners, based on self-reported data collected retrospectively. By contrast, this thesis is based on a critical ethnographic case study focusing on pedagogic process. Analysing data from participant observation, discussions, interviews, and students’ learning journals, I hone in on the micro-level functions of power, space, and place in shaping not only what is taught and learned during the BB programme, but also how, where and why this happens. Suspended in the tension between Bourdieu’s theory of social and cultural reproduction (1990) and Curry-Stevens’ post-Freirean ‘Pedagogy for the Privileged’ (2007), I principally use Bernstein’s notion of ‘pedagogic device’ (2000) to analyse how programme Instructors counter-intentionally facilitated socially reproductive, rather than transformative, learning. I argue that the programme reproduces social inequalities by enabling privileged people to accumulate a specific form of ‘cultural capital’ (Bourdieu, 1977) – cross-cultural transformation capital (CCTC). This is gathered by gaining supposedly “authentic” knowledge through “real” experiences with “the Other” in culturally “pure” spaces, accessible only to “travellers” and uncontaminated by “tourists”. I show how this creates patterns of pedagogic segregation whereby specific types of pedagogic space produce specific types of knowledge. However, paradoxically, I also describe sporadic, unpredictable pockets of transformative learning in which students engage critically with their privileged positioning in asymmetric power structures. I thus contend that (socially) transformative pedagogic space is constituted in complex, contradictory ways, but also by pedagogy that must connect personal and social change. I conclude that greater attention to power and space is critical to transformative pedagogic theory and practice which can be framed and conceptualised in spatial terms, as the crossing and reconfiguring of boundaries

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops

Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies

Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes.

The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a non-polar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies