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Biomedical subjects

Lori A Rowe

Publications and source records attributed to Lori A Rowe.

4 recordsLinked to original sources
PubMed2005

The standardization of radiation skin care in British Columbia: a collaborative approach.

PURPOSE/OBJECTIVES: To develop evidence-based practice guidelines for and standardize the care of radiation skin reactions. DATA SOURCES: Peer-reviewed scientific journals and texts and a survey of the guidelines in use at leading cancer treatment facilities in Canada, the United States, the United Kingdom, and Australia. DATA SYNTHESIS: A formal reference document with recommended guidelines was developed. Consensus was obtained from all relevant disciplines, and the guidelines were implemented successfully into practice. CONCLUSIONS: The document introduced a major change in practice from the maintenance of a dry radiation treatment area to the promotion of skin cleanliness and hydration, as well as the adoption of the principles of moist wound healing. Annual review indicated that dissemination of (94%) and compliance with (78%) the guidelines were good. IMPLICATIONS FOR NURSING: The process to develop, obtain consensus for, and implement evidence-based practice guidelines was an exemplary demonstration of teamwork and interdisciplinary collaboration.

British Columbia↗
PubMed2005

Whole plasmid mutagenic PCR for directed protein evolution.

Protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). Optimization of protein expression is essential for the development of sensitive and precise high throughput assays. Here we optimize the performance of a plasmid-borne Escherichia coli lacZ gene in two rounds of directed evolution. First, its promoter was "randomized" by whole plasmid polymerase chain reaction (PCR) and intra-molecular self-ligation. A genetically stable constitutive expression vector was isolated in an in vivo genetic selection. Second, the entire plasmid was randomly mutated in a slightly mutagenic long polymerase chain reaction. The PCR products were digested with a restriction enzyme, self-ligated by T4 DNA ligase and transformed into E. coli. The resulting library of beta-galactosidase (beta-gal) mutants consisted mostly ( approximately 80%) of hypomorphs, suggesting that the mutation rate was appropriate for directed evolution applications. We isolated and characterized 14 variants with increased activity in reactions with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). The purified protein derived from one clone exhibited a 100-fold improvement in k(cat) over its parent in reactions with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal). This latter result clearly demonstrates the utility of whole plasmid mutagenic PCR for directed protein evolution.

Cloning, Molecular↗
PubMed2003

A comparison of directed evolution approaches using the beta-glucuronidase model system.

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.

Directed Molecular Evolution↗