Screening for Cytochrome P450 Reactivity by Harnessing Catalase as Reporter Enzyme

The big screen : We have devised a high‐throughput screening method for organic peroxide‐dependent P450 reactivity by taking advantage of a previously undescribed activity of catalase, which was used as reporter enzyme. This two‐step assay, followed by liquid chromatography/mass spectrometry analyses, allowed the facile identification of several new substrates for bacterial P450 enzymes.

     

Coupling Interrupted Fischer and Multicomponent Joullié-Ugi to Chase Chemical Diversity: from Batch to Sustainable Flow Synthesis of Peptidomimetics

The generation of peptidomimetic substructures for medicinal chemistry purposes requires effective and divergent synthetic methods. We present in this work an efficient flow process that allows quick modulation of reagents for Joullié-Ugi multicomponent reaction, using spiroindolenines as core motifs. This sterically hindered imine equivalent could successfully be diversified using various isocyanides and amino acids in generally good space-time yields. A telescoped flow process combining interrupted Fischer reaction for spiroindolenine synthesis and subsequent Joullié-Ugi-type modification resulted in product formation in very good overall yield in less than 2 hours compared to 48 hours required in batch mode. The developed protocol can be seen as a general tool for rapid and facile generation of peptidomimetic compounds. We also showcase preliminary biological assessments for the prepared compounds.     

Refining Genotypes and Phenotypes in KCNA2-Related Neurological Disorders

Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel K_v1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of-function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)–valine(406)–proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype–phenotype correlation, variability, and predicted functional impact of KCNA2 variants.     

Viscous and Aeroelastic Effects on Wind Turbine Blades. The VISCEL project. Part I: 3D Navier–Stokes Rotor simulations

Aerodynamic modelling of HAWT rotors by means of “engineering methods” has reached a saddle point, where no further development can be expected without a breakthrough in understanding the physics of unsteady, rotating three‐dimensional flows. However, such a breakthrough becomes ever more necessary, as the size of the wind turbines increases. With the experimental work in that direction being mostly limited to observing the phenomena and interpreting the associated mechanisms, and its increased cost, alternatives are being sought. The use of CFD techniques and state‐of‐the‐art Navier–Stokes solvers is considered a very serious contender, a belief shared by the members of the present consortium, which has worked on the VISCEL JOR3‐CT98‐0208 Joule III project. This project's goal was to determine the aerodynamic characteristics as well as the aeroelastic behaviour of wind turbine blades across their broad range of operational conditions, from attached to highly separated flow regimes. The work programme included specific tasks for the validation and assessment of existing 3D solvers, for the parametric study of 3D flow around realistic blades and for the investigation of aeroelastic stability, at the blade section level. Copyright © 2003 John Wiley & Sons, Ltd.     

Disambiguating Visual Motion by Form-Motion Interaction—a Computational Model

The neural mechanisms underlying motion segregation and integration still remain unclear to a large extent. Local motion estimates often are ambiguous in the lack of form features, such as corners or junctions. Furthermore, even in the presence of such features, local motion estimates may be wrong if they were generated near occlusions or from transparent objects. Here, a neural model of visual motion processing is presented that involves early stages of the cortical dorsal and ventral pathways. We investigate the computational mechanisms of V1-MT feedforward and feedback processing in the perception of coherent shape motion. In particular, we demonstrate how modulatory MT-V1 feedback helps to stabilize localized feature signals at, e.g. corners, and to disambiguate initial flow estimates that signal ambiguous movement due to the aperture problem for single shapes. In cluttered environments with multiple moving objects partial occlusions may occur which, in turn, generate erroneous motion signals at points of overlapping form. Intrinsic-extrinsic region boundaries are indicated by local T-junctions of possibly any orientation and spatial configuration. Such junctions generate strong localized feature tracking signals that inject erroneous motion directions into the integration process. We describe a simple local mechanism of excitatory form-motion interaction that modifies spurious motion cues at T-junctions. In concert with local competitive-cooperative mechanisms of the motion pathway the motion signals are subsequently segregated into coherent representations of moving shapes. Computer simulations demonstrate the competency of the proposed neural model.     

Kinetic masks: a new approach and device for dispersing biologically relevant fluids

The controlled dispersion of fluids, particularly biologically relevant solutions in micro-volumes, is of high practical interest in biotechnology and medicine. Pharmaceutical test assays, for example, need a method for the fast and defined deposition of fluid samples. Most current micro-dispensing methods, i.e. contact-based pin printing, have problems such as time delays, limited dosing velocity, minimum volume or high interference that limit biological applications. Spraying techniques suffer from a lack of reproducibility; a defined deposition of samples on targets is not possible. Here, we introduce a new method for the parallel and spatially defined dispersion of many micro-volumes that overcomes disadvantages of common micro-dispensers. The overall approach is that a fluid drop, produced by a droplet generator, falls on a free trajectory with a defined kinetic energy, and is split by a masking unit placed perpendicular to the flight direction into at least two smaller droplets (Zimmermann et al. in Method and device for dosing fluid media, WO/2002/102515, Germany, 2002). On the target, the resulting droplets form reproducible patterns, which are enlarged and scalable images of the grid pattern. Possible applications for this method are non-contact cell patterning, cell encapsulation, cryopreservation and fast mixing processes in micro-volumes. Here, we use this method for the direct and defined parallel positioning of cell suspensions on specific substrates, which can be useful for test assays, tissue engineering and cryopreservation.