Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-alpha/beta1, and CAS, a member of the importin-beta family, which exports importin-alpha to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-alpha, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-alpha in the nuclei of growing cells and in vitro. Importin-alpha generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-alpha, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-alpha in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.

BackgroundSignaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.Methodology/Principal FindingsTo better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.Conclusions/SignificanceOur studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.

In procedural-based medicine, the technical ability can be a critical determinant of patient outcomes. Psychomotor performance occurs in real-time, hence a continuous assessment is necessary to provide action-oriented feedback and error avoidance guidance. We outline a deep learning application, the Intelligent Continuous Expertise Monitoring System (ICEMS), to assess surgical bimanual performance at 0.2-s intervals. A long-short term memory network was built using neurosurgeon and student performance in 156 virtually simulated tumor resection tasks. Algorithm predictive ability was tested separately on 144 procedures by scoring the performance of neurosurgical trainees who are at different training stages. The ICEMS successfully differentiated between neurosurgeons, senior trainees, junior trainees, and students. Trainee average performance score correlated with the year of training in neurosurgery. Furthermore, coaching and risk assessment for critical metrics were demonstrated. This work presents a comprehensive technical skill monitoring system with predictive validation throughout surgical residency training, with the ability to detect errors.

BACKGROUND: The methodology of assessment and training of surgical skills is evolving to deal with the emergence of competency-based training. Artificial neural networks (ANNs), a branch of artificial intelligence, can use newly generated metrics not only for assessment performance but also to quantitate individual metric importance and provide new insights into surgical expertise. OBJECTIVE: To outline the educational utility of using an ANN in the assessment and quantitation of surgical expertise. A virtual reality vertebral osteophyte removal during a simulated surgical spine procedure is used as a model to outline this methodology. METHODS: Twenty-one participants performed a simulated anterior cervical diskectomy and fusion on the Sim-Ortho virtual reality simulator. Participants were divided into 3 groups, including 9 postresidents, 5 senior residents, and 7 junior residents. Data were retrieved from the osteophyte removal component of the scenario, which involved using a simulated burr. The data were manipulated to initially generate 83 performance metrics spanning 3 categories (safety, efficiency, and motion) of which only the most relevant metrics were used to train and test the ANN. RESULTS: The ANN model was trained on 6 safety metrics to a testing accuracy of 83.3%. The contributions of these performance metrics to expertise were revealed through connection weight products and outlined 2 identifiable learning patterns of technical skills. CONCLUSION: This study outlines the potential utility of ANNs which allows a deeper understanding of the composites of surgical expertise and may contribute to the paradigm shift toward competency-based surgical training.