We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47C but not at 55C and above. Variants that were kanamycin resistant at 63°C were obtained by selection of spontaneous mutants, by passage ofa shuttle plasmid through theEscherichia colimutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the frt variant by selecting for kanamycin resistance at 70C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.The ability of some microorganisms to grow at extreme temperatures (1) implies that their enzymes are stable and active at these temperatures. This is largely borne out when enzymes from thermophilic sources are studied in vitro; such enzymes are indeed more thermostable than the equivalents isolated from phylogenetically related mesophilic organisms (2). Correlations between an increase in the proportion of hydrophobic residues and the degree of thermostability have been observed (3-5). Internal electrostatic interactions (6) and disulfide linkages (7) have also been proposed as features that stabilize proteins. However, the role of individual amino acid residues in enhancing the resistance to thermal denaturation of an enzyme from a thermophile is not known.We wish to understand the contributions of individual amino acids to the overall stability of a protein's structure. Comparisons between enzymes from mesophiles and thermophiles are complicated because these proteins, although homologous, usually differ in more than one residue. More precise inferences can be based on comparisons of temperature-sensitive mutations which encode, as a result of single amino acid changes, proteins that retain activity but are less resistant to heat denaturation than the wild-type counterpart. Thus, for example, many temperature-sensitive mutations of phage T4 lysozyme have been studied with the aim of correlating the changes in stability with changes in the protein structure (8). However, an x-ray crystallographic study of one of these mutant enzymes (9) revealed that a variant in which a histidine residue repla...
Video games have become the most dominant and successful entertainment industry worldwide; however, many video game development (VGD) projects and studios struggle to succeed. At present, there are no commonly accepted VGD best practices or frameworks that can bring together the complex and competing needs of software engineering and creative production. Although studios are reportedly using agile frameworks, the actual extent of application and effectiveness of agile practices in the VGD context is unclear. Therefore, the aim of this study is to empirically determine how and why agile frameworks are applied in VGD. Semi-structured interviews were conducted with eight New Zealand VGD studios. It was found that the agile frameworks, Scrum and Kanban, must often be adapted from their conventional use to meet the needs of different pipelines and delivery milestones within the phases of VGD. However, it seems that not all the needs of VGD can be met by current agile frameworks. Furthermore, inexperience with agile practices often leads to misunderstanding and misimplementing them in ways that seem to contribute to commonly experienced collaboration challenges.
In these perspectives, we share the experiences of eight cochlear implant (CI) recipients who are musicians, and their efforts within and outside of audiological appointments to achieve satisfying music experiences. Their experiences were previously shared in a panel discussion as part of the 3rd Music and Cochlear Implant Symposium hosted at The University of Cambridge, United Kingdom. Following the symposium, the panel members and moderator developed and completed a follow-up questionnaire to facilitate a formal analysis of the following questions: (a) What forms of support for optimizing music exist within clinical CI appointments, including counseling, mapping, assessment, and rehabilitation? (b) What forms of support do CI users who are interested in music desire? (c) What self-initiated approaches can be used to improve music perception, enjoyment, and participation? Using qualitative methodology, the questionnaire data were coded, aggregated into themes, and then into core categories. The primary themes that emerged from the data were (a) limited levels of support for optimizing music outcomes within normal clinical appointments, (b) difficulties in current mapping and assessment in relation to music perception, and (c) limited availability of clinically sponsored training/rehabilitation for music. These CI recipients then recommended clinical protocol changes and described self-initiated rehabilitation. These findings were examined in relation to literature on clinical practices for CI users, auditory rehabilitation, and patient-centered care, emphasizing best practices and barriers to audiological care. The data as related to healthcare trends were conceptualized and developed into a proposed Reciprocal Model for Music Rehabilitation (RMMR).
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