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     

Recent trends in early outcome of adult patients after heart transplantation: a single-institution review of 251 transplants using standard donor organs.

Older age, prior transplantation, pulmonary hypertension, and mechanical support are commonly seen in current potential cardiac transplant recipients. Transplants in 436 consecutive adult patients from 1994 to 1999 were reviewed. There were 251 using standard donors in 243 patients (age range 18-69 years). To emphasize recipient risk, 185 patients who received a nonstandard donor were excluded from analysis. The indications for transplant were ischemic heart disease (n = 123, 47%), dilated cardiomyopathy (n = 82, 32%), and others (n=56, 21%). One hundred and forty-nine (57%) recipients were listed as status I; 5 and 6% were supported with an intra-aortic balloon and an assist device, respectively. The 30-d survival and survival to discharge were 94.7 and 92.7%, respectively; 1-year survival was 89.1%. Causes of early death were graft failure (n = 6), infection (n = 4), stroke (n = 4), multiorgan failure (n = 3) and rejection (n = 2). Predictors were balloon pump use alone (OR= 11.4, p =0.002), pulmonary vascular resistance > 4 Wood units (OR = 5.7, p = 0.007), pretransplant creatinine > 2.0 mg/dL (OR = 6.9, p = 0.004) and female donor (OR = 8.3, p = 0.002). Recipient age and previous surgery did not affect short-term survival. Heart transplantation in the current era consistently offers excellent early and 1-year survival for well-selected recipients receiving standard donors. Early mortality tends to reflect graft failure while hospital mortality may be more indicative of recipient selection.     

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.     

High tolerance of lampreys to Kepone® toxicity

Larval sea lampreys (Petromyzon marinus) were exposed to the organochlorine insecticide Kepone^® in freshwater solution in a continuous flow diluter system at 12 and 20°C. At 12°, the 36-hr LC50, 96-hr LC50, and incipient lethal concentrations were 1,100, 444 and 145 g Kepone/ L, respectively, while at 20°, the 96-hr LC50 was 414 g/L. These are the highest LC50 values for Kepone ever reported for a fish species. Rates at which larval lampreys accumulate and clear Kepone were measured at 12°C. The depuration rate constant (K_d: 0.13–0.46 per day) was the highest ever reported in a fish species, so rapid elimination may contribute to the exceptional ability of lampreys to survive acute Kepone poisoning. The uptake rate constant (K_u) was 450–650 per day, and the bioconcentration factor averaged about 1900. The most likely source of high tolerance of lampreys to Kepone is an ability to withstand high tissue levels: Lampreys survived body burdens of 500–600 g Kepone/g, exceeding all other known vertebrates. Technical difficulties associated with the use of Kepone solutions are discussed, such as precipitation and loss from solution through apparent volatilization.     

Aspects of three dimensional reconstruction for a multi ring positron tomography

An important feature of multi ring positron tomographs is the inter plane septa, the purpose of which is to reduce random and scattered coincidences. In general, such septa also eliminate the coincidence lines of response between pairs of detectors more than one ring apart. The operation of a camera without septa must result in an increase not only in the true coincidence rate, but also in the singles, and therefore in the dead time and randoms rate, and in the scattered coincidences. A configuration option in the coincidence hardware of the 8 ring, 15 slice ECAT 931/08-12 enables a full set of 64 sinograms to be acquired when the septa are removed. The detector normalisation and transmission data for studies with the septa out can be obtained using a rotating pin source. To take maximum advantage of the additional signal, the emission data must be reconstructed using a fully three dimensional reconstruction algorithm. This paper presents an analysis of some phantom studies acquired without septa and reconstructed in three dimensions. The results are compared with data acquired with septa for the same phantoms imaged under similar conditions. It is found that, with the septa removed, the signal to noise for a uniform, 20 cm diameter cylinder improves by a factor of 2.8 in the centre of the field of view, whereas in regions distant from the centre in the axial direction, the signal to noise decreases due to the increase in scatter and randoms. An improvement in signal to noise is observed in 6 cm of the 10 cm axial length of the tomograph.This article was presented at the 1st EEC workshop on accuracy determination in PET, January 19–20th. 1989 Pisa, Italy (COMAC-BME Concerted Project Characterization and Standardization of PET Instrumentation)     

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.