Biomedical subjects

Robert Keenan

Publications and source records attributed to Robert Keenan.

5 recordsLinked to original sources

PubMed2005

Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy.

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.

Acetyltransferases↗

PubMed2005

Video-assisted wedge resection and local radiotherapy for peripheral lung cancer in high-risk patients: the Cancer and Leukemia Group B (CALGB) 9335, a phase II, multi-institutional cooperative group study.

OBJECTIVES: This study examined the feasibility of thoracoscopic wedge resection and radiotherapy for clinical T1 lesions in patients with compromised cardiopulmonary status. METHODS: In this phase II, prospective, multicenter, cooperative group trial, high-risk patients had one or more of the following risk factors: forced expiratory volume in 1 second less than 40%, carbon monoxide diffusing capacity in lung less than 50%, and maximum oxygen consumption less than 45 mm Hg. Patients underwent video-assisted wedge resection followed by local (56 Gy) radiotherapy. The primary end point was the proportion of patients whose disease could be completely resected and who received radiotherapy without treatment complications. RESULTS: Between September 1995 and September 1999, a total of 65 patients were accrued, of which 58 were eligible (52% male, median age 69 years). Pathologic staging resulted in upgrading to T2 or greater in 16 of 58 cases (28%) and in reassessment as benign in 10 of 58 cases (17%). Conversion to thoracotomy was required in 10 cases (17%), including 1 of 10 benign T1-size lesion (10%), 4 of 35 non-small cell lung cancer T1 lesions (13%), and 5 of 14 non-small cell lung cancer T2 lesions (31%). Resection margins were positive in 5 patients: 6% of T1 and 23% of T2. Surgery was aborted in 2 cases (3.5%), and operative mortality was 4%. Overall operative failure rates of video-assisted wedge resection were 20% for benign T1-size lesions, 22% for T1 non-small cell lung cancer, 21% for all T1 lesions, 50% for T2 non-small cell lung cancer, and 29% for all lesions in this study (clinical T1). Prolonged air leaks occurred in 10%, pneumonia in 6%, and respiratory failure in 4%. Thirty-one patients were eligible for radiotherapy; 3 of them refused, and 1 died before treatment. Among the 28 patients who received radiotherapy, severe dyspnea was noted in 3 patients (11%) and moderate pneumonitis in 4 (14%). CONCLUSIONS: Clinical staging in high-risk patients is often inaccurate (45% difference from pathologic staging). Intention to treat clinically staged T1 disease by video-assisted wedge resection is associated with a high failure rate. Pathologically staged T1 lesions can be successfully resected in 75% of cases; however, narrow resection margins remain a concern.

Aged↗

PubMed2005

Laboratory-directed protein evolution.

Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to "evolve" in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

DNA Shuffling↗

PubMed2004

Clinical outcome of laparoscopic antireflux surgery for patients with irritable bowel syndrome.

BACKGROUND: The prevalence of irritable bowel syndrome (IBS) is higher among subjects with gastroesophageal reflux disease (GERD). This study aimed to assess the effect of IBS on the postoperative outcome of antireflux surgery. METHODS: For this study, 102 patients who underwent laparoscopic fundoplication were screened preoperatively for IBS with the Rome II criteria. There were 32 patients in the IBS group and 70 patients in the non-IBS group. Most of the patients (97%) (31 of 32 IBS and 68 of 70 non-IBS patients) had both pre- and postoperative IBS evaluation. A visual analog GERD-specific scoring scale was used to evaluate GERD symptoms prospectively. RESULTS: In both groups, GERD symptom scores were statistically improved postoperatively. Of the 31 IBS patients 25 (80.6%) showed a reduction in their symptoms below the Rome II criteria for IBS diagnosis postoperatively. CONCLUSION: Irritable bowel syndrome does not have a negative effect on the outcome of laparoscopic antireflux surgery. Surgical correction of GERD may improve the severity of irritable bowel symptoms.

Adult↗

PubMed2002

Influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients.

BACKGROUND: Although influenza and parainfluenza viruses commonly cause respiratory tract infections in the community, their incidence and clinical implications in adult lung transplant recipients have received little attention. METHODS: We performed a retrospective cohort study of influenza and parainfluenza viral infections in adult lung transplant recipients at the University of Pittsburgh Medical Center. RESULTS: Between January 1989 and March 1999, 39 cases (single-lung 25, double-lung 14) of influenza or parainfluenza respiratory viral infection were identified at a mean of 1.7 years (SD+/-1.4) after transplantation. The mean length of admission was 7 days. The cases included 15 patients with influenza (A, 11; B, 4) and 24 with parainfluenza (para1, 7; para2, 2; para3, 15). The median age at diagnosis was 48 years; there were 19 females and 20 males. Symptoms were reported in 30 patients and lasted for a median of 7 days before admission. These included cough (64%), shortness of breath (56%), and temperature elevation (33%). Chest infiltrates were seen in 14 (36%) patients, and 5 (13%) of them required intubation and mechanical ventilation. Viral pneumonia was diagnosed in 10 (5 influenza and 5 parainfluenza) patients, and concurrent bacterial pneumonia occurred in 4 patients. Transbronchial biopsy was performed in 36 patients, of whom 23 (64%) showed some degree of acute allograft rejection. CONCLUSION: Influenza and parainfluenza respiratory viral infections are associated with significant morbidity in adult lung transplant recipients. Active vaccination programs and the development of new antiviral agents active against these viruses are important for prevention.

Adult↗