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Rescue of a single yeast artificial chromosome from a cotransformation event utilizing segregation at meiosis
During the construction of yeast artificial chromosome (YAC) libraries to facilitate mapping of the human genome, two YACs may be cotransformed into the same yeast cell, making further analysis very difficult. We present a simple method to rescue the required YAC that utilizes the segregation of chromosomes at meiosis. In brief, we crossed the cotransformed yeast cell with a non-YAC-containing strain and induced the resulting diploid to sporulate and undergo meiosis. The new haploid generation included some yeast cells that contained only the desired YAC. These YACs were analyzed by conventional methods. To exclude the possibility that major rearrangement occurred during the procedure, we analyzed the YACs with restriction enzymes that cut only rarely. We conclude that this is a useful technique to rescue cotransformed YACs. © 1993.
Molecular and cellular correlates of human nerve regeneration: ADCYAP1 encoding PACAP enhances sensory neuron outgrowth
Abstract We only have a rudimentary understanding of the molecular and cellular determinants of human nerve regeneration. Here, we use carpal tunnel syndrome (CTS) as a human model system to prospectively evaluate correlates of neural regeneration and their relationship with clinical recovery after decompression surgery. At 6 months post-surgery, we noted a significant improvement of median nerve neurophysiological and somatosensory function. Serial skin biopsies revealed a partial recovery of intraepidermal innervation, whose extent correlated with symptom improvement. In myelinated afferents, nodal length increased postoperatively. Transcriptional profiling of the skin revealed 23 differentially expressed genes following decompression, with ADCYAP1 (encoding PACAP) being the most strongly upregulated and showing an association with regeneration of intraepidermal nerve fibres. Using human induced pluripotent stem cell-derived sensory neurons, we confirmed that PACAP significantly enhances axon outgrowth in vivo . Since PACAP signals through G-protein receptors, this pathway provides an interesting therapeutic target for human sensory nerve regeneration.
Assessing the long-term effectiveness of interferon-beta and glatiramer acetate in multiple sclerosis: final 10-year results from the UK multiple sclerosis risk-sharing scheme
<jats:sec><jats:title>Background</jats:title><jats:p>Because multiple sclerosis (MS) is a chronic disease causing disability over decades, it is crucial to know if the short-term effects of disease-modifying therapies reported in randomised controlled trials reduce long-term disability. This 10-year prospective observational study of disability outcomes (Expanded Disability Status Scale (EDSS) and utility) was set up, in conjunction with a risk-sharing agreement between payers and producers, to investigate this issue.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The outcomes of the UK treated patients were compared with a modelled untreated control based on the British Columbia MS data set to assess the long-term effectiveness of these treatments. Two complementary analysis models were used: a multilevel model (MLM) and a continuous Markov model.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>4862 patients with MS were eligible for the primary analysis (mean and median follow-up times 8.7 and 10 years). EDSS worsening was reduced by 28% (MLM), 7% (Markov) and 24% time-adjusted Markov in the total cohort, and by 31% (MLM) and 14% (Markov) for relapsing remitting patients. The utility worsening was reduced by 23%–24% in the total cohort and by 24%–31% in the RR patients depending on the model used. All sensitivity analyses showed a treatment effect. There was a 4-year (CI 2.7 to 5.3) delay to EDSS 6.0. An apparent waning of treatment effect with time was seen. Subgroup analyses suggested better treatment effects in those treated earlier and with lower EDSS scores.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>This study supports a beneficial effect on long-term disability with first-line MS disease-modifying treatments, which is clinically meaningful. However the waning effect noted requires further study.</jats:p></jats:sec>
BACKGROUND: Although hypothermia is widely used to protect the brain during cardiac and neurologic surgery, the optimal level of cooling has not been established. This study examined the protective effect of graded levels of surface cooling on cerebral function in rats after complete global cerebral ischemia. METHODS: Groups of ketamine-anesthetized rats (13 animals in each group) were cooled to cranial temperatures of 34, 30, 27, 24, or 22 degrees C before circulatory arrest. Also a normothermic (37 degrees C) group was tested. After cooling, an 11-min circulatory arrest was produced by atraumatic chest compression. Circulatory arrest was followed by cardiopulmonary resuscitation and rewarming without postischemic intensive care. On the fifth postinsult day, neurologic outcome was scored on a 50-point neurodeficit scale (NDS 0 = normal). The percent of ischemic pyramidal neurons in the CA1 hippocampal region was also determined. RESULTS: There were no survivors in the normothermic group. Neurologic recovery was enhanced with 30 degrees C cranial temperature, as compared to outcome in the 34 degrees C group. Further cooling did not change outcome. The neurodeficit scales were significantly lower in all other groups compared to the 34 degrees C group on the fifth postinsult day. The percent of ischemic neurons did not change significantly as a function of cooling, but the lowest count appeared at 27 degrees C. CONCLUSION: In this model, moderate (30 degrees C) cooling improved neurologic outcome. There was no additional benefit from more extreme hypothermia.
Introduction: Attentional focus narrows as individuals concentrate on tasks. Missing an event that would otherwise appear obvious is termed a perceptual error. These forms of perceptual failure are well-recognised in psychological literature, but little attention has been paid to them in medicine. Cognitive workload and expertise modulate risk, although how these factors interplay in practice is unclear. This video-based experiment was designed to explore the hypothesis that perceptual errors affect clinicians. Methods: 142 volunteers with varying levels of experience of adult resuscitation were shown a short video depicting a simulated cardiac arrest. This video included a series of change-events designed to elicit perceptual errors. The experiment was conducted on-line, with participants watching the video and then responding via combinations of open-ended free-text and directed questioning. Results: 141 people experienced at least a single perceptual error. Even the most clinically significant event (disconnection of the patient's oxygen supply) was missed by three in four viewers. Although expertise was associated with increased likelihood of detecting an occurrence, even highly significant events were missed by up to two thirds of the most experienced observers. Discussion: This study demonstrates, for the first time, that perceptual errors occur during healthcare-relevant scenarios at significant levels. Events such as an oxygen malfunction would meaningfully affect patient outcome and, although expertise conferred some advantages, events were still missed more often than not. Data acquisition is fundamental to good-quality situational awareness. These results suggest perceptual error may be a contributor to adverse events in practice. © 2014 Elsevier Ireland Ltd.