Swelling and mechanical properties of alginate hydrogels with respect to promotion of neural growth

Soft alginate hydrogels support robust neurite outgrowth, but their rapid disintegration in solutions of high ionic strength restricts them from long-term in vivo applications. Aiming to enhance the mechanical stability of soft alginate hydrogels, we investigated how changes in pH and ionic strength during gelation influence the swelling, stiffness, and disintegration of a three-dimensional (3D) alginate matrix and its ability to support neurite outgrowth. Hydrogels were generated from dry alginate layers through ionic crosslinks with Ca(2+) (<=10 mM) in solutions of low or high ionic strength and at pH 5.5 or 7.4. High- and low-viscosity alginates with different molecular compositions demonstrated pH and ionic strength-independent increases in hydrogel volume with decreases in Ca(2+) concentrations from 10 to 2 mM. Only soft hydrogels that were synthesized in the presence of 150 mM of NaCl (Ca-alginateNaCl) displayed long-term volume stability in buffered physiological saline, whereas analogous hydrogels generated in NaCl-free conditions (Ca-alginate) collapsed. The stiffnesses of Ca-alginateNaCl hydrogels elevated from 0.01 to 19 kPa as the Ca(2+)-concentration was raised from 2 to 10 mM; however, only Ca-alginateNaCl hydrogels with an elastic modulus <=1.5 kPa that were generated with <=4 mM of Ca(2+) supported robust neurite outgrowth in primary neuronal cultures. In conclusion, soft Ca-alginateNaCl hydrogels combine mechanical stability in solutions of high ionic strength with the ability to support neural growth and could be useful as 3D implants for neural regeneration in vivo

Single-cell, whole-embryo phenotyping of mammalian developmental disorders

Mouse models are a critical tool for studying human diseases, particularly developmental disorders. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution

Ein Gassystem für die äußeren Spurkammern von HERA-B

Das Standardmodell zur Beschreibung der Materie und der in ihr herrschenden Kräfte weist einige Lücken auf. Das Experiment HERA-B soll nach dem theoretischen nun auch den experimentellen Beweis dafür liefern, warum Materie und Antimaterie nach dem Urknall nicht in gleichen Anteilen im Universum vorzufinden ist. Es wird im folgenden ein Gassystem für eine Detektorkomponente von HERA-B, dem "Äußeren Spurkammersystem" beschrieben. Um die Funktion dieses Gasdetektors aufrecht zu erhalten, müssen die 26 Module an ein Gassystem angeschlossen sein, welches in jedem Modul einen Volumenaustausch des Detektorgases pro Stunde und einen Differenzdruck gegenüber der Atmosphäre von (0 ± 0,5) mbar gewährleistet. Um zu bestimmen, wie ein solches Gassystem ausgelegt werden muß, und ob es möglich ist, mehrere Module mit einer Druckregulation zu betreiben, wurde ein Testgassystem aufgebaut, welches die Bedingungen von HERA-B simulieren soll

Autism-associated mutations in the Ca-v beta(2) calcium-channel subunit increase Ba2+-currents and lead to differential modulation by the RGK-protein Gem

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of Ca-v beta(2) subunits of VGCCs. From this, we derived the hypothesis that several Ca-v beta(2)-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a Ca-v beta(2d) backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba2+ currents carried by Ca(v)1.2 pore subunits co-transfected with the previously described Ca-v beta(2) mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated Ca-v beta(2) subunits with the RGK protein Gem was analyzed by coimmunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All Ca-v beta(2) mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: Ca-v beta(2d_G167S) > Ca-v beta(2d_v2D) > Ca-v beta(2d_S197F). In conclusion, patch-clamp recordings of ASD-associated Ca-v beta(2d) mutations revealed differential modulation of Ba2+ currents carried by Ca(v)1.2 suggesting kind of an electrophysiological fingerprint each. The increase in current finally observed with all Ca-v beta(2d) mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism