Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation assays

A novel technique for the selection of mutated DNA sequences,termed mismatch cleavage-polymerase chain reaction (MC-PCR),is proposed. The method is based on hybridizing genomic DNAwith a suitable probe, several 100 bp long. Mutated DNA sequenceswill form mismatched heteroduplexes which are cleaved by usingresolvases. Cleaved heteroduplexes are detected by ligationto an oligonucleotide adaptor and then amplified by using PCR.If practical, this technique would have considerable advantagesover the restriction site mutation (RSM) method. Failure toachieve cleavage efficiencies of close to 100% will not compromisesuccess. This is because positive signals (PCR amplification)arise from cleaved mutated sites and not, as in RSM, from DNAsequences resistant to cleavage by restriction endonucleases.Furthermore, the mutational target is much larger than in RSM.It would be possible to screen stretches of DNA several 100bp in length for mutations. Any mutation, independent of itslocation, could be identified. The usefulness of MC-PCR forthe genotypic selection of mutants will depend on the effectivenesswith which a small number of mismatched heteroduplexes can berecognized, cleaved and ligated. ¹To whom correspondence should be addressed     

Newspaper coverage of residential fires: an opportunity for prevention communication.

BACKGROUND: Worldwide, fire-related burns are a major cause of unintentional injury, morbidity and mortality, with the majority of deaths occurring in developing countries. In the US, as in other countries, most fatal fires occur in the home. Effective prevention strategies for residential fires are, however, currently underutilized. The news media is one available communication channel to promote such strategies, and analyzing current news coverage is a first step towards incorporating media advocacy into injury prevention efforts related to residential fires. METHODS: Four daily newspapers circulating widely in Maryland were monitored for 1 year. Articles describing residential fires were coded for measures of prominence, content and frame. Analysis focused on measures of issue newsworthiness, reporting of causation and consequences of fires, and inclusion of public health context and conveyance of prevention messages. RESULTS: The data indicate that fires are newsworthy, with 374 relevant news articles in a 1-year period, 32% of which appear on the first page of a section. Coverage generally concerned recent local fire events. Most articles discussed the consequences of fires (88%), and identified a causal factor (58%). Only 36%, however, included prevention information, and less than one-quarter set residential fires in a public health context. CONCLUSION: The newsworthiness of residential fires provides a clear opportunity for widespread communication around injury prevention. Improving media advocacy will entail framing discussion of recent fire events in such a way as to support inclusion of prevention strategies and a public health context in news coverage.     

Morphometric analysis of the great vessels in early fetal life

Pathological examination of the heart and great vessels wasperformed in 61 specimens obtained after surgical terminationof pregnancy for psychosocial indications at 9–18 weeksof gestation. The aorta and pulmonary trunk were identifiedand external diameters were measured at the level of, and distalto the aortic valve and pulmonary valve, the level of the aorticisthmus and thoracic aorta, and the proximal and distal ductusarteriosus. All eight vessel diameters increased linearly withgestational age and the ratio of the diameter of the aorticisthmus to that of the aortic valve or the distal ductus arteriosusalso increased with gestation. Early pregnancy is characterizedby rapid growth of the fetal head and this may well be the consequenceof a preferential distribution of left ventricular output infavour of the head due to relative narrowing of the aortic isthmusat this gestation.     

Care at the end of life: a novel curriculum module implemented by medical students.

End-of-life (EOL) and palliative care education in medical school curricula stand at a crossroads. Consensus has emerged that these topics merit systematic instruction throughout medical school training, yet curricula all too often consist of sporadic lectures focused on bioethics instead of clinical skills. The medical student authors identified a deficit in their curriculum, and designed and implemented an EOL curriculum module for their colleagues. In early 2000 the authors surveyed senior medical students about their experiences with EOL and palliative education, identifying deficits in clinical training and recommendations for interventions. They then designed a case-based educational module to teach EOL communication skills to medical students commencing clinical training. Faculty with national and local experience with EOL and palliative care reviewed the curriculum. Twelve of these faculty were oriented to the curriculum and then taught it in pairs to groups of 12 to 16 medical students in 2000 and 2001. The curriculum develops skills, attitudes, and knowledge relevant for communicating bad news and establishing treatment options in the EOL setting by utilizing trigger videos, group discussion, role plays, and case discussions. Approximately 75% of the 86 eligible students attended the module in 2000 and 2001. Feedback has guided the curriculum's refinement by the medical student authors. In addition, a standardized patient exercise, introduced in 2001, allowed students to reinforce the skills learned during the module.     

Effect of a new synthetic serum replacement on insulin and somatostatin secretion from isolated rat pancreatic islets in long-term culture

Summary Serum contains insulin degrading components. We have evaluated the insulin and somatostatin secretion from isolated rat pancreatic islets during a 2-wk culture period using three different serum-containing media, and one serum-free medium with a synthetic serum replacement. Islets incubated in serum-free medium elicited significantly higher daily insulin and somatostatin secretions than islets incubated in the serum-containing media. After a 2-wk culture period, islets from the serum-free medium secreted significantly more insulin and somatostatin than islets cultured in other media when stimulated with 25 mmol/liter glucose together with 15 mmol/liter theophylline. We conclude that the serum-free medium is superior for long-term culture of rat pancreatic islets.     

Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli

Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell. Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway. Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins. Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step. Here we present a method--premature targeting--for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway. We fused FtsQ to the FtsZ-binding protein ZapA such that FtsQ was targeted to FtsZ rings independently of proteins FtsA and FtsK, which are normally required for FtsQ localization. Analysis of the resulting ZapA-FtsQ fusion suggests that FtsQ associates with a large complex of cell division proteins and that premature targeting of FtsQ can restore localization of this complex under conditions in which neither FtsQ nor the associated proteins would normally be localized.