Reflektometrische Interferenzspektroskopie (RIfS) zur Detektion ganzer Legionella pneumophila-Zellen und Entwicklung regenerierbarer funktionaler Oberflächen für Anwendungen in der Biosensorik.

Verlässliche und effiziente Methoden der Legionellen-Detektion sind für den Bereich der Hygiene in öffentlichen Gebäuden überaus wichtig. Die Legionellose ist eine der häufigsten Todesursachen infolge von nosokomial erworbenen Infektionen. Die etablierten Legionellentests liefern erst nach 3-7 Tagen Ergebnisse. Eine schnellere Methode wird dringend gebraucht. Beispielsweise werden Biosensoren in vielen Bereichen der Analytik eingesetzt. Allerdings lässt sich auf den entsprechenden Sensorchips nur jeweils ein Experiment durchführen. Diese Einschränkung kostet Material und Geld. Die RIfS erweist sich als Methode, mit der ganze Legionella pneumophila-Zellen nachgewiesen werden können. Allerdings liegt die untere Nachweisgrenze bei 1000000-10000000 Zellen/ml. Eine Quantifizierung der Zellen erweist sich als schwierig, weil die Sensorsignale am Ende der Dissoziationsphase nicht zuverlässig mit den Zell-Konzentrationen korrelierten. Der Grund ist offenbar, dass die Zellen auf der OF keine im physikalischen Sinne wohl definierte Schicht bilden. Durch REM konnten große leere Bereiche auf einer OF festgestellt werden und häufige Überlappungen der Zellen. Die Korrelation zwischen der Steigung in der Anfangsphase der Bindung und der Konzentration ist offenbar zuverlässiger, da Überlappungen etc. in der Anfangsphase keine große Rolle spielen

Worker churn in the cross section and over time: New evidence from Germany

Worker churn is procyclical in the German labor market. We study the plant-level connection of churn and employment growth using the new Administrative Wage and Labor Market Flow Panel from 1975 to 2014. Churn is V-shaped in employment growth. Through analyzing this pattern by worker skill, age, and tenure, we establish that churn is unlikely to result from plant reorganization but rather from uncertainty about match quality. In a dynamic labor demand framework with a time-to-hire friction, churn can be interpreted as a manifestation of idiosyncratically stochastic separation shocks. These shocks become larger and more predictable during booms, leading to procyclical churn.The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FTP/2007-2013) / ERC Grant agreement no. 282740. Felix Wellschmied gratefully acknowledges support from the Spanish Ministry of Economics through research grants ECO2014-56384-P, MDM 2014-0431, and Comunidad de Madrid MadEco-CM (S2015/HUM-3444). Heiko Stüber gratefully acknowledge support from the German Research Foundation (DFG) under priority program “The German Labor Market in a Globalized World” (SPP 1764). Christian Merkl gratefully acknowledge support from SPP 1764 and the Hans Frisch Stiftung

Gene silencing pathways found in the green alga Volvox carteri reveal insights into evolution and origins of small RNA systems in plants

Background: Volvox carteri (V. carteri) is a multicellular green alga used as model system for the evolution of multicellularity. So far, the contribution of small RNA pathways to these phenomena is not understood. Thus, we have sequenced V. carteri Argonaute 3 (VcAGO3)-associated small RNAs from different developmental stages. Results: Using this functional approach, we define the Volvox microRNA (miRNA) repertoire and show that miRNAs are not conserved in the closely related unicellular alga Chlamydomonas reinhardtii. Furthermore, we find that miRNAs are differentially expressed during different life stages of V. carteri. In addition to miRNAs, transposon-associated small RNAs or phased siRNA loci, which are common in higher land plants, are highly abundant in Volvox as well. Transposons not only give rise to miRNAs and other small RNAs, they are also targets of small RNAs. Conclusion: Our analyses reveal a surprisingly complex small RNA network in Volvox as elaborate as in higher land plants. At least the identified VcAGO3-associated miRNAs are not conserved in C. reinhardtii suggesting fast evolution of small RNA systems. Thus, distinct small RNAs may contribute to multicellularity and also division of labor in reproductive and somatic cells

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization

Reflektometrische Interferenzspektroskopie (RIfS) zur Detektion ganzer Legionella pneumophila-Zellen und Entwicklung regenerierbarer funktionaler Oberflächen für Anwendungen in der Biosensorik.

Verlässliche und effiziente Methoden der Legionellen-Detektion sind für den Bereich der Hygiene in öffentlichen Gebäuden überaus wichtig. Die Legionellose ist eine der häufigsten Todesursachen infolge von nosokomial erworbenen Infektionen. Die etablierten Legionellentests liefern erst nach 3-7 Tagen Ergebnisse. Eine schnellere Methode wird dringend gebraucht. Beispielsweise werden Biosensoren in vielen Bereichen der Analytik eingesetzt. Allerdings lässt sich auf den entsprechenden Sensorchips nur jeweils ein Experiment durchführen. Diese Einschränkung kostet Material und Geld. Die RIfS erweist sich als Methode, mit der ganze Legionella pneumophila-Zellen nachgewiesen werden können. Allerdings liegt die untere Nachweisgrenze bei 1000000-10000000 Zellen/ml. Eine Quantifizierung der Zellen erweist sich als schwierig, weil die Sensorsignale am Ende der Dissoziationsphase nicht zuverlässig mit den Zell-Konzentrationen korrelierten. Der Grund ist offenbar, dass die Zellen auf der OF keine im physikalischen Sinne wohl definierte Schicht bilden. Durch REM konnten große leere Bereiche auf einer OF festgestellt werden und häufige Überlappungen der Zellen. Die Korrelation zwischen der Steigung in der Anfangsphase der Bindung und der Konzentration ist offenbar zuverlässiger, da Überlappungen etc. in der Anfangsphase keine große Rolle spielen

Sequence and functional differences in the ATPase domains of CHD3 and SNF2H promise potential for selective regulability and drugability

Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q‐ and K‐residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild‐type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of CHD3 is sufficient for nucleosome remodeling. Together with the ADP data, our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q‐ and K‐residues distinctly regulate ATPase‐dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics

The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis

Several long non-coding RNAs (lncRNAs) act as regulators of cellular homeostasis; however, few of these molecules were functionally characterized in a mature human tissue environment. Here, we report that the lncRNA LINC00941 is a crucial regulator of human epidermal homeostasis. LINC00941 is enriched in progenitor keratinocytes and acts as a repressor of keratinocyte differentiation. Furthermore, LINC00941 represses SPRR5, a previously uncharacterized molecule, which functions as an essential positive regulator of keratinocyte differentiation. Interestingly, 54.8% of genes repressed in SPRR5-deficient epidermal tissue are induced in LINC00941-depleted organotypic epidermis, suggesting a common mode of action for both molecules

Magnet for the Needle in Haystack: “Crystal Structure First” Fragment Hits Unlock Active Chemical Matter Using Targeted Exploration of Vast Chemical Spaces

Fragment-based drug discovery (FBDD) has successfully led to approved therapeutics for challenging and “undruggable” targets. In the context of FBDD, we introduce a novel, multidisciplinary method to identify active molecules from purchasable chemical space. Starting from four small-molecule fragment complexes of protein kinase A (PKA), a template-based docking screen using Enamine’s multibillion REAL Space was performed. A total of 93 molecules out of 106 selected compounds were successfully synthesized. Forty compounds were active in at least one validation assay with the most active follow-up having a 13,500-fold gain in affinity. Crystal structures for six of the most promising binders were rapidly obtained, verifying the binding mode. The overall success rate for this novel fragment-to-hit approach was 40%, accomplished in only 9 weeks. The results challenge the established fragment prescreening paradigm since the standard industrial filters for fragment hit identification in a thermal shift assay would have missed the initial fragments