Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material

Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5%concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detectio

Collapse of turbulence in stably stratified channel flow: a transient phenomenon

The collapse of turbulence in a pressure-driven, cooled channel flow is studied by using 3D direct numerical simulations (DNS) in combination with theoretical analysis using a local similarity model. Previous studies with DNS reported a definite collapse of turbulence in cases when the normalized surface cooling h/L (with h the channel depth and L the Obukhov length) exceeded a value of 0.5. A recent study by the present authors succeeded in explaining this collapse using the so-called maximum sustainable heat flux (MSHF) theory. This states that collapse may occur when the ambient momentum of the flow is too weak to transport enough heat downward to compensate for the surface cooling. The MSHF theory predicts that, in pressure-driven flows, acceleration of the fluid after collapse will eventually cause a regeneration of turbulence, in contrast with the aforementioned DNS results. It also predicts that the flow should be able to survive ‘supercritical’ cooling rates, in cases when sufficient momentum is applied to the initial state. Here, both predictions are confirmed using DNS simulations. It is also shown that in DNS a recovery of turbulence will occur naturally, provided that perturbations of finite amplitude are imposed on the laminarized state and provided that sufficient time for flow acceleration is allowed. As such, we conclude that the collapse of turbulence in this configuration is a temporary, transient phenomenon for which a universal cooling rate does not exist. Finally, in the present work a one-to-one comparison between a parametrized, local similarity model and the turbulence-resolving model (DNS) is made. Although local similarity originates from observations that represent much larger Reynolds numbers than those covered by our DNS simulations, both methods appear to predict very similar mean velocity (and temperature) profiles. This suggests that in-depth analysis with DNS can be an attractive complementary tool with which to study atmospheric physics, in addition to tools that are able to represent high Reynolds number flows like large-eddy simulations

Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior

<p>Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.</p>