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.     

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission “on” signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.     

Numerical characterization and experimental verification of an in-plane MEMS-actuator with thin-film aluminum heater

In this paper, a novel concept of a thermo-mechanical MEMS actuator using aluminum thin-film heaters on a thermal oxide for electrical insulation is presented. The actuator is part of an universal tensile testing platform for thermo-mechanical material characterization of one dimensional materials on a micro- and nano-scopic scale under different environmental conditions, as varying temperatures, pressure, moisture or even vacuum and is realised in BDRIE technology. It is shown, that the actuator concept fulfills the requirements for the use in a tensile loading stage along with heterogeneously integrated nanofunctional elements, following a specimen centered approach in line with bottom-up self-assembly processes. Simulation and experiment agree very well in the thermal and mechanical domain and allow subsequent optimisation of the actuator performance.     

Aqueous heterophase polymerization of styrene—a study by means of multi-angle laser light scattering

An online multi-angle laser light scattering study of ab initio surfactant-free styrene emulsion polymerization reveals unexpected results regarding the development of the dispersity during the whole reaction starting from mixing styrene and water at reaction temperature. The experimentally observed change in the dispersity, which is an indirect measure of the average characteristic size of the colloidal objects, allows the identification of three characteristic intervals. During interval A the equilibration of styrene in water is characterized by the formation of styrene domains, which increase in number and size until an equilibrium state is reached. This means that there is virtually no homogeneous/molecular styrene in water solution but rather nanodroplets of about 200 nm in diameter (assuming spherical shape) are formed. During interval B, after initiation of the polymerization and particle formation, the dispersity increases again as the average size decreases and the number of scattering objects increases. The polymer particles suck up the monomer from the monomer nanodroplets, which leads to the disappearance of the nanodroplets and to a decrease in the average size. During interval C the average size increases again due to the predominantly growth of the polystyrene particles.     

New inhibitors of the Tat-TAR RNA interaction found with a "fuzzy" pharmacophore model

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.     

On stability of signature-based similarity measures for content-based image retrieval

Retrieving similar images from large image databases is a challenging task for today’s content-based retrieval systems. Aiming at high retrieval performance, these systems frequently capture the user’s notion of similarity through expressive image models and adaptive similarity measures. On the query side, image models can significantly differ in quality compared to those stored on the database side. Thus, similarity measures have to be robust against these individual quality changes in order to maintain high retrieval performance. In this paper, we investigate the robustness of the family of signature-based similarity measures in the context of content-based image retrieval. To this end, we introduce the generic concept of average precision stability, which measures the stability of a similarity measure with respect to changes in quality between the query and database side. In addition to the mathematical definition of average precision stability, we include a performance evaluation of the major signature-based similarity measures focusing on their stability with respect to querying image databases by examples of varying quality. Our performance evaluation on recent benchmark image databases reveals that the highest retrieval performance does not necessarily coincide with the highest stability.     

Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors

The size of the inner water cavity of reversed micelles formedin a triple system ‘water-surfactant-organic solvent’can be widely varied by changing the degree of surfactant hydration.This gives grounds to use reversed micelles as matrix microreactorsfor the design of supramolecular complexes of proteins. Usingultracentrifugation analysis, it has been demonstrated thatthe oligomeric composition of various enzymes (ketoglutaratedehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase) solubilized in reversed micelles of Aerosol OT[sodium bis(2-ethylehexyl)sulfosuccinate] in octane changesupon variation of the degree of hydration. An oligomeric complexforms under conditions when the radius of the micelle innercavity is big enough to incorporate this complex as a whole.At lower degrees of hydration the micelles ‘uncouple’such complexes to their components. The catalytic propertiesof various oligomeric complexes have been studied. Possibilitiesof using reversed micelles for the separation of subunits ofoligomeric enzymes under non-denaturating conditions have beendemonstrated. In particular, the isolated subunits of alkalinephosphatase, lactic dehydrogenase and glyceralde-hyde-3-phosphatedehydrogenase have been found to be active in Aerosol OT reversedmicelles. The dependences of the catalytic activity of oligomericenzymes represent saw-like curves. The maxima of the catalyticactivity observed at these curves relate to the functioningof various oligomeric forms of an enzyme. The radii of the micelleinner cavity under conditions when these maxima are observedcorrelate with the linear dimensions of the enzyme oligomericforms. Correlation of the position of a maximum with the shapeof an oligomeric complex is discussed.     

Towards language-to-language transformation

This paper proposes a simplicity-oriented approach and framework for language-to-language transformation of, in particular, graphical languages. Key to simplicity is the decomposition of the transformation specification into sub-rule systems that separately specify purpose-specific aspects. We illustrate this approach by employing a variation of Plotkin’s Structural Operational Semantics (SOS) for pattern-based transformations of typed graphs in order to address the aspect ‘computation’ in a graph rewriting fashion. Key to our approach are two generalizations of Plotkin’s structural rules: the use of graph patterns as the matching concept in the rules, and the introduction of node and edge types. Types do not only allow one to easily distinguish between different kinds of dependencies, like control, data, and priority, but may also be used to define a hierarchical layering structure. The resulting Type-based Structural Operational Semantics (TSOS) supports a well-structured and intuitive specification and realization of semantically involved language-to-language transformations adequate for the generation of purpose-specific views or input formats for certain tools, like, e.g., model checkers. A comparison with the general-purpose transformation frameworks ATL and Groove, illustrates along the educational setting of our graphical WebStory language that TSOS provides quite a flexible format for the definition of a family of purpose-specific transformation languages that are easy to use and come with clear guarantees.

     

Extended bisection method for parameter identification of the transient heat conduction equation for thermo-elastic deformations during drilling

A new interdisciplinary approach is discribed to identifying unknown parameters using an extended version of the known interval bisection method. This developed method is based on the use of finite elements for calibrating the simulation calculation. The resulting thermo-elastic deformations which occur in drilling processes with impaired cooling lubrication are to be used as correction values for tool positioning in the NC control. Based on the strong impact on workpiece temperature of machining, a simulation approach is presented for calculating the temperature fields and their thermo-elastic consequences. In addition, methods are presented to correct these effects. This paper particularly deals with the temperature fields of drilling operations. Special attention is paid to the technique employed for iterative numerical determination of the unknown heat flux η _w and heat transfer coefficient \(\bar {\gamma }\) values. Finally, the data obtained from experiments are compared with those achieved by numerical simulation in order to verify the efficiency of simulation and determination of parameters.     

“From microtiter plates to droplets” tools for micro-fluidic droplet processing

Droplet-based microfluidic allows high throughput experimentation in with low volume droplets. Essential fluidic process steps are on the one hand the proper control of the droplet composition and on the other hand the droplet processing, manipulation and storage. Beside integrated fluidic chips, standard PTFE-tubings and fluid connectors can be used in combination with appropriate pumps to realize almost all necessary fluidic processes. The segmented flow technique usually operates with droplets of about 100–500 nL volume. These droplets are embedded in an immiscible fluid and confined by channel walls. For the integration of segmented flow applications in established research workflows—which are usually base on microtiter plates—robotic interface tools for parallel/serial and serial/parallel transfer operations are necessary. Especially dose–response experiments are well suited for the segmented flow technique. We developed different transfer tools including an automated “gradient take-up tool” for the generation of segment sequences with gradually changing composition of the individual droplets. The general working principles are introduced and the fluidic characterizations are given. As exemplary application for a dose–response experiment the inhibitory effect of antibiotic tetracycline on Escherichia coli bacteria cultivated inside nanoliter droplets was investigated.