Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

The expression of a new histone variant H3.Y increases during cellular stress to regulate cell cycle progression and gene expression

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture

Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins

Fusion of Bacterial Flagellin to a Dendritic Cell-Targeting αCD40 Antibody Construct Coupled With Viral or Leukemia-Specific Antigens Enhances Dendritic Cell Maturation and Activates Peptide-Responsive T Cells

Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors in vivo with antibody–antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of in vivo targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality in vitro and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an αCD40 antibody containing a CMV pp65-derived peptide as an antigen domain (αCD40CMV) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.FlgCMV). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40CMV highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40CMV alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.FlgCMV induced significantly higher CMVNLV-specific T cell activation and proliferation compared to αCD40CMV in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-γ/TNF-α+ CD8+ T cells and 3.9-fold increase in % CMVNLV-specific dextramer+ CD8+ T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of αCD40mNPM1 and αCD40.FlgmNPM1 antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.FlgmNPM1-loaded DCs more potently activated allogeneic mNPM1CLA-specific T cells compared to αCD40mNPM1. These in vitro results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of αCD40.FlgmNPM1 after targeting of dendritic cells in vivo using AML xenograft models

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

The human genome forms thousands of “contact domains”, which are intervals of enhanced contact frequency. Some, called “loop domains” are thought to form by cohesin-mediated loop extrusion. Others, called “compartmental domains”, form due to the segregation of active and inactive chromatin into A and B compartments. Recently, Hi-C studies revealed that the depletion of cohesin leads to the disappearance of all loop domains within a few hours, but strengthens compartment structure. Here, we combine live cell microscopy, super-resolution microscopy, Hi-C, and studies of replication timing to examine the longer-term consequences of cohesin degradation in HCT-116 human colorectal carcinoma cells, tracking cells for up to 30 hours. Surprisingly, cohesin depleted cells proceed through an aberrant mitosis, yielding a single postmitotic cell with a multilobulated nucleus. Hi-C reveals the continued disappearance of loop domains, whereas A and B compartments are maintained. In line with Hi-C, microscopic observations demonstrate the reconstitution of chromosome territories and chromatin domains. An interchromatin channel system (IC) expands between chromatin domain clusters and carries splicing speckles. The IC is lined by active chromatin enriched for RNA Pol II and depleted in H3K27me3. Moreover, the cells exhibit typical early-, mid-, and late- DNA replication timing patterns. Our observations indicate that the functional nuclear compartmentalization can be maintained in cohesin depleted pre- and postmitotic cells. However, we find that replication foci – sites of active DNA synthesis – become physically larger consistent with a model where cohesin dependent loop extrusion tends to compact intervals of replicating chromatin, whereas their genomic boundaries are associated with compartmentalization, and do not change.3D FISH3D fluorescence in situ hybridization3D SIM3D structured illumination microscopyAIDauxin inducible degronANC / INCactive / inactive nuclear compartmentCTchromosome territoryCD(C)chromatin domain (cluster)CTCFCCCTC binding factorDAPI4’,6-diamidino-2-phenylindoleEdU5-Ethynyl-2’-deoxyuridineHi-Cchromosome conformation capturing combined with deep sequencingICinterchromatin compartmentMLNmultilobulated nucleusNCnucleosome clusterPBSphosphate buffered salinePBSTphosphate buffered saline with 0.02% TweenPRperichromatin regionRDreplication domainRLreplication labelingTADtopologically associating domai

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity

Roadmap Batterie-Produktionsmittel 2030. Update 2016

Die VDMA Roadmap Batterie-Produktionsmittel 2030 wurde 2016 vollständig aktualisiert, überarbeitet und um wichtige Aspekte ergänzt. Sie befasst sich mit prozesstechnischen Herausforderungen, die wesentlich zum Fortschritt in der industriellen Fertigung von Li-Ionen-Batterien für den Einsatz in der Elektromobilität und in der stationären Energiespeicherung beitragen. Für 16 Kernbereichen werden notwendige Technologiedurchbrüche identifiziert und der entsprechende Forschungsbedarf sowie perspektivische Lösungsansätze bis 2030 aufgezeigt