Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases

Proposal for a Small Scheme Implementation

Scheme is a lexically scoped dialect of LISP developed at MIT. In this report we determine the feasibility of implementing a Scheme-based programming/application environment on a contemporary personal computer such as the Apple Macintosh. The absense of virtual memmory, coupled with a limitation on the maximum amount of physical memory, means that space is at a premium. We suggest the use of bytecodes and sketch a possible instruction set. Because of space constraints, tail-recursion optimization and an efficient mechanism for the reclamation of inaccessible contexts are also examined. Using the built-in operating system and user interface of the Macintosh realizes speed, functionality, and friendliness but raises a number of interesting issues. For example, the Pascal and assembler routines make many assumptions about data representation, type checking, and parameter passing. Since an implementation of Scheme is likely to have radically different conventions, the two environments must be interfaced smoothly and efficiently. In addition to the bytecode instruction set, we specify the virtual machine informally, discuss the implementation of basic and advanced features, and estimate the performance of such an implementation, and finally evaluate the proposed design

Reduction in adhesive small-bowel obstruction by Seprafilm® adhesion barrier after intestinal resection

Contains fulltext : 50472.pdf (publisher's version ) (Closed access)INTRODUCTION: Although Seprafilm has been demonstrated to reduce adhesion formation, it is not known whether its usage would translate into a reduction in adhesive small-bowel obstruction. METHODS: This was a prospective, randomized, multicenter, multinational, single-blind, controlled study. This report focuses on those patients who underwent intestinal resection (n = 1,701). Before closure of the abdomen, patients were randomized to receive Seprafilm or no treatment. Seprafilm was applied to adhesiogenic tissues throughout the abdomen. The incidence and type of bowel obstruction was compared between the two groups. Time to first adhesive small-bowel obstruction was compared during the course of the study by using survival analysis methods. The mean follow-up time for the occurrence of adhesive small-bowel obstruction was 3.5 years. RESULTS: There was no difference between the treatment and control group in overall rate of bowel obstruction. The incidence of adhesive small-bowel obstruction requiring reoperation was significantly lower for Seprafilm patients compared with no-treatment patients: 1.8 vs. 3.4 percent (P < 0.05). This finding represents an absolute reduction in adhesive small-bowel obstruction requiring reoperation of 1.6 percent and a relative reduction of 47 percent. In addition, a stepwise multivariate analysis indicated that the use of Seprafilm was the only predictive factor for reducing adhesive small-bowel obstruction requiring reoperation. In both groups, 50 percent of first adhesive small-bowel obstruction episodes occurred within 6 months after the initial surgery with nearly 30 percent occurring within the first 30 days. Additionally no first adhesive small-bowel obstruction events were reported in Years 4 and 5 of follow-up. CONCLUSIONS: The overall bowel obstruction rate was unchanged; however, adhesive small-bowel obstruction requiring reoperation was significantly reduced by the use of Seprafilm, which was the only factor that predicted this outcome

Perfusion Assessment in Left-Sided/Low Anterior Resection (PILLAR III): A Randomized, Controlled, Parallel, Multicenter Study Assessing Perfusion Outcomes With PINPOINT Near-Infrared Fluorescence Imaging in Low Anterior Resection.

BackgroundIndocyanine green fluoroscopy has been shown to improve anastomotic leak rates in early phase trials.ObjectiveWe hypothesized that the use of fluoroscopy to ensure anastomotic perfusion may decrease anastomotic leak after low anterior resection.DesignWe performed a 1:1 randomized controlled parallel study. Recruitment of 450 to 1000 patients was planned over 2 years.SettingsThis was a multicenter trial.PatientsIncluded patients were those undergoing resection defined as anastomosis within 10 cm of the anal verge.InterventionPatients underwent standard evaluation of tissue perfusion versus standard in conjunction with perfusion evaluation using indocyanine green fluoroscopy.Main outcome measuresPrimary outcome was anastomotic leak, with secondary outcomes of perfusion assessment and the rate of postoperative abscess requiring intervention.ResultsThis study was concluded early because of decreasing accrual rates. A total of 25 centers recruited 347 patients, of whom 178 were randomly assigned to perfusion and 169 to standard. The groups had comparable tumor-specific and patient-specific demographics. Neoadjuvant chemoradiation was performed in 63.5% of perfusion and 65.7% of standard (p &gt; 0.05). Mean level of anastomosis was 5.2 ± 3.1 cm in perfusion compared with 5.2 ± 3.3 cm in standard (p &gt; 0.05). Sufficient visualization of perfusion was reported in 95.4% of patients in the perfusion group. Postoperative abscess requiring surgical management was reported in 5.7% of perfusion and 4.2% of standard (p = 0.75). Anastomotic leak was reported in 9.0% of perfusion compared with 9.6% of standard (p = 0.37). On multivariate regression analysis, there was no difference in anastomotic leak rates between perfusion and standard (OR = 0.845 (95% CI, 0.375-1.905); p = 0.34).LimitationsThe predetermined sample size to adequately reduce the risk of type II error was not achieved.ConclusionsSuccessful visualization of perfusion can be achieved with indocyanine green fluoroscopy. However, no difference in anastomotic leak rates was observed between patients who underwent perfusion assessment versus standard surgical technique. In experienced hands, the addition of routine indocyanine green fluoroscopy to standard practice adds no evident clinical benefit. See Video Abstract at http://links.lww.com/DCR/B560.Valoracin de la irrigacin de lado izquierdo/reseccin anterior baja pilar iii un estudio aleatorizado, controlado, paralelo y multicntrico que evala los resultados de la irrigacin con pinpoint imgenes de fluorescencia cercana al infrarrojo en la reseccin anterior bajaANTECEDENTES:Se ha demostrado que la fluoroscopia con verde de indocianina mejora las tasas de fuga anastomótica en ensayos en fases iniciales.OBJETIVO:Nuestra hipótesis es que la utilización de fluoroscopia para asegurar la irrigación anastomótica puede disminuir la fuga anastomótica luego de una resección anterior baja.DISEÑO:Realizamos un estudio paralelo, controlado, aleatorizado 1:1. Se planificó el reclutamiento de 450-1000 pacientes durante 2 años.AMBITO:Multicéntrico.PACIENTES:Pacientes sometidos a resección definida como una anastomosis dentro de los 10cm del margen anal.INTERVENCIÓN:Pacientes que se sometieron a la evaluación estándar de la irrigación tisular contra la estándar en conjunto con la valoración de la irrigación mediante fluoroscopia con verde indocianina.PRINCIPALES VARIABLES EVALUADAS:El principal resultado fue la fuga anastomótica, y los resultados secundarios fueron la evaluación de la perfusión y la tasa de absceso posoperatorio que requirió intervención.RESULTADOS:Este estudio se cerró anticipadamente debido a la disminución de las tasas de acumulación. Un total de 25 centros reclutaron a 347 pacientes, de los cuales 178 fueron, de manera aleatoria, asignados a perfusión y 169 a estándar. Los grupos tenían datos demográficos específicos del tumor y del paciente similares. Recibieron quimio-radioterapia neoadyuvante el 63,5% de la perfusión y el 65,7% del estándar (p&gt; 0,05). La anastomosis estuvo en un nivel promedio de 5,2 + 3,1 cm en perfusión en comparación con 5,2 + 3,3 cm en estándar (p&gt; 0,05). Se reportó una visualización suficiente de la perfusión en el 95,4% de los pacientes del grupo de perfusión. El absceso posoperatorio que requirió tratamiento quirúrgico fue de 5,7% de los perfusion y en el 4,2% del estándar (p = 0,75). Se informó fuga anastomótica en el 9,0% de la perfusión en comparación con el 9,6% del estándar (p = 0,37). En el análisis de regresión multivariante, no hubo diferencias en las tasas de fuga anastomótica entre la perfusión y el estándar (OR 0,845; IC del 95% (0,375; 1,905); p = 0,34).LIMITACIONES:No se logró el tamaño de muestra predeterminado para reducir satisfactoriamente el riesgo de error tipo II.CONCLUSIÓN:Se puede obtener una visualización adecuada de la perfusión con ICG-F. Sin embargo, no se observaron diferencias en las tasas de fuga anastomótica entre los pacientes que se sometieron a evaluación de la perfusión versus la técnica quirúrgica estándar. En manos expertas, agregar ICG-F a la rutina de la práctica estándar no agrega ningún beneficio clínico evidente. Consulte Video Resumen en http://links.lww.com/DCR/B560. (Traducción-Dr Juan Antonio Villanueva-Herrero)