Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Decision-to-incision times and maternal and infant outcomes.

Journal ArticleOBJECTIVE: To measure decision-to-incision intervals and related maternal and neonatal outcomes in a cohort of women undergoing emergency cesarean deliveries at multiple university-based hospitals comprising the National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. METHODS: All women undergoing a primary cesarean delivery at a Network center during a 2-year time span were prospectively ascertained. Emergency procedures were defined as those performed for umbilical cord prolapse, placental abruption, placenta previa with hemorrhage, nonreassuring fetal heart rate pattern, or uterine rupture. Detailed information regarding maternal and neonatal outcomes, including the interval from the decision time to perform cesarean delivery to the actual skin incision, was collected. RESULTS: Of the 11,481 primary cesarean deliveries, 2,808 were performed for an emergency indication. Of these, 1,814 (65%) began within 30 minutes of the decision to operate. Maternal complication rates, including endometritis, wound infection, and operative injury, were not related to the decision-to-incision interval. Measures of newborn compromise including umbilical artery pH less than 7 and intubation in the delivery room were significantly greater when the cesarean delivery was commenced within 30 minutes, likely attesting to the need for expedited delivery. Of the infants with indications for an emergency cesarean delivery who were delivered more than 30 minutes after the decision to operate, 95% did not experience a measure of newborn compromise. CONCLUSION: Approximately one third of primary cesarean deliveries performed for emergency indications are commenced more than 30 minutes after the decision to operate, and the majority were for nonreassuring heart rate tracings. In these cases, adverse neonatal outcomes were not increased. LEVEL OF EVIDENCE: II-2

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

Assessment of higher order cognitive skills in undergraduate education: modified essay or multiple choice questions? Research paper

Background: Reliable and valid written tests of higher cognitive function are difficult to produce, particularly for the assessment of clinical problem solving. Modified Essay Questions (MEQs) are often used to assess these higher order abilities in preference to other forms of assessment, including multiple-choice questions (MCQs). MEQs often form a vital component of end-of-course assessments in higher education. It is not clear how effectively these questions assess higher order cognitive skills. This study was designed to assess the effectiveness of the MEQ to measure higher-order cognitive skills in an undergraduate institution. Methods: An analysis of multiple-choice questions and modified essay questions (MEQs) used for summative assessment in a clinical undergraduate curriculum was undertaken. A total of 50 MCQs and 139 stages of MEQs were examined, which came from three exams run over two years. The effectiveness of the questions was determined by two assessors and was defined by the questions ability to measure higher cognitive skills, as determined by a modification of Bloom's taxonomy, and its quality as determined by the presence of item writing flaws. Results: Over 50% of all of the MEQs tested factual recall. This was similar to the percentage of MCQs testing factual recall. The modified essay question failed in its role of consistently assessing higher cognitive skills whereas the MCQ frequently tested more than mere recall of knowledge. Conclusion: Construction of MEQs, which will assess higher order cognitive skills cannot be assumed to be a simple task. Well-constructed MCQs should be considered a satisfactory replacement for MEQs if the MEQs cannot be designed to adequately test higher order skills. Such MCQs are capable of withstanding the intellectual and statistical scrutiny imposed by a high stakes exit examination.Edward J Palmer, Peter G Devit

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

Three little pieces for computer and relativity

Numerical relativity has made big strides over the last decade. A number of problems that have plagued the field for years have now been mostly solved. This progress has transformed numerical relativity into a powerful tool to explore fundamental problems in physics and astrophysics, and I present here three representative examples. These "three little pieces" reflect a personal choice and describe work that I am particularly familiar with. However, many more examples could be made.Comment: 42 pages, 11 figures. Plenary talk at "Relativity and Gravitation: 100 Years after Einstein in Prague", June 25 - 29, 2012, Prague, Czech Republic. To appear in the Proceedings (Edition Open Access). Collects results appeared in journal articles [72,73, 122-124

Exploring health systems research and its influence on policy processes in low income countries

<p>Abstract</p> <p>Background</p> <p>The interface between research and policymaking in low-income countries is highly complex. The ability of health systems research to influence policy processes in such settings face numerous challenges. Successful analysis of the research-policy interface in these settings requires understanding of contextual factors as well as key influences on the interface. <it>Future Health Systems (FHS): Innovations for Equity </it>is a consortium conducting research in six countries in Asia and Africa. One of the three cross-country research themes of the consortium is analysis of the relationship between research (evidence) and policy making, especially their impact on the poor; insights gained in the initial conceptual phase of FHS activities can inform the global knowledge pool on this subject.</p> <p>Discussion</p> <p>This paper provides a review of the research-policy interface in low-income countries and proposes a conceptual framework, followed by directions for empirical approaches. First, four developmental perspectives are considered: social institutional factors; virtual versus grassroots realities; science-society relationships; and construction of social arrangements. Building on these developmental perspectives three research-policy interface entry points are identified: 1. Recognizing policy as complex processes; 2. Engaging key stakeholders: decision-makers, providers, scientists, and communities; and 3. Enhancing accountability. A conceptual framework with three entry points to the research-policy interface – policy processes; stakeholder interests, values, and power; and accountability – within a context provided by four developmental perspectives is proposed. Potential empirical approaches to the research-policy interface are then reviewed. Finally, the value of such innovative empirical analysis is considered.</p> <p>Conclusion</p> <p>The purpose of this paper is to provide the background, conceptual framework, and key research directions for empirical activities focused on the research-policy interface in low income settings. The interface can be strengthened through such analysis leading to potential improvements in population health in low-income settings. Health system development cognizant of the myriad factors at the research-policy interface can form the basis for innovative future health systems.</p

Impairment of Auditory-Motor Timing and Compensatory Reorganization after Ventral Premotor Cortex Stimulation

Integrating auditory and motor information often requires precise timing as in speech and music. In humans, the position of the ventral premotor cortex (PMv) in the dorsal auditory stream renders this area a node for auditory-motor integration. Yet, it remains unknown whether the PMv is critical for auditory-motor timing and which activity increases help to preserve task performance following its disruption. 16 healthy volunteers participated in two sessions with fMRI measured at baseline and following rTMS (rTMS) of either the left PMv or a control region. Subjects synchronized left or right finger tapping to sub-second beat rates of auditory rhythms in the experimental task, and produced self-paced tapping during spectrally matched auditory stimuli in the control task. Left PMv rTMS impaired auditory-motor synchronization accuracy in the first sub-block following stimulation (p<0.01, Bonferroni corrected), but spared motor timing and attention to task. Task-related activity increased in the homologue right PMv, but did not predict the behavioral effect of rTMS. In contrast, anterior midline cerebellum revealed most pronounced activity increase in less impaired subjects. The present findings suggest a critical role of the left PMv in feed-forward computations enabling accurate auditory-motor timing, which can be compensated by activity modulations in the cerebellum, but not in the homologue region contralateral to stimulation

A theory of organizational readiness for change

<p>Abstract</p> <p>Background</p> <p>Change management experts have emphasized the importance of establishing organizational readiness for change and recommended various strategies for creating it. Although the advice seems reasonable, the scientific basis for it is limited. Unlike individual readiness for change, organizational readiness for change has not been subject to extensive theoretical development or empirical study. In this article, I conceptually define organizational readiness for change and develop a theory of its determinants and outcomes. I focus on the organizational level of analysis because many promising approaches to improving healthcare delivery entail collective behavior change in the form of systems redesign--that is, multiple, simultaneous changes in staffing, work flow, decision making, communication, and reward systems.</p> <p>Discussion</p> <p>Organizational readiness for change is a multi-level, multi-faceted construct. As an organization-level construct, readiness for change refers to organizational members' shared resolve to implement a change (change commitment) and shared belief in their collective capability to do so (change efficacy). Organizational readiness for change varies as a function of how much organizational members value the change and how favorably they appraise three key determinants of implementation capability: task demands, resource availability, and situational factors. When organizational readiness for change is high, organizational members are more likely to initiate change, exert greater effort, exhibit greater persistence, and display more cooperative behavior. The result is more effective implementation.</p> <p>Summary</p> <p>The theory described in this article treats organizational readiness as a shared psychological state in which organizational members feel committed to implementing an organizational change and confident in their collective abilities to do so. This way of thinking about organizational readiness is best suited for examining organizational changes where collective behavior change is necessary in order to effectively implement the change and, in some instances, for the change to produce anticipated benefits. Testing the theory would require further measurement development and careful sampling decisions. The theory offers a means of reconciling the structural and psychological views of organizational readiness found in the literature. Further, the theory suggests the possibility that the strategies that change management experts recommend are equifinal. That is, there is no 'one best way' to increase organizational readiness for change.</p