Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.     

Effect of welding parameters on the heat-affected zone of AISI409 ferritic stainless steel

One of the main problems during the welding of ferritic stainless steels is severe grain growth within the heat-affected zone (HAZ). In the present study, the microstructural characteristics of tungsten inert gas (TIG) welded AISI409 ferritic stainless steel were investigated by electron backscattered diffraction (EBSD), and the effects of welding parameters on the grain size, local misorientation, and low-angle grain boundaries were studied. A 3-D finite element model (FEM) was developed to predict the effects of welding parameters on the holding time of the HAZ above the critical temperature of grain growth. It is found that the base metal is not fully recrystallized. During the welding, complete recrystallization is followed by severe grain growth. A decrease in the number of low-angle grain boundaries is observed within the HAZ. FEM results show that the final state of residual strains is caused by competition between welding plastic strains and their release by recrystallization. Still, the decisive factor for grain growth is heat input.     

MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer

Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.     

Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.     

Genetic deficiency of tartrate-resistant acid phosphatase associated with skeletal dysplasia, cerebral calcifications and autoimmunity

Vertebral and metaphyseal dysplasia, spasticity with cerebral calcifications, and strong predisposition to autoimmune diseases are the hallmarks of the genetic disorder spondyloenchondrodysplasia. We mapped a locus in five consanguineous families to chromosome 19p13 and identified mutations in ACP5, which encodes tartrate-resistant phosphatase (TRAP), in 14 affected individuals and showed that these mutations abolish enzyme function in the serum and cells of affected individuals. Phosphorylated osteopontin, a protein involved in bone reabsorption and in immune regulation, accumulates in serum, urine and cells cultured from TRAP-deficient individuals. Case-derived dendritic cells exhibit an altered cytokine profile and are more potent than matched control cells in stimulating allogeneic T cell proliferation in mixed lymphocyte reactions. These findings shed new light on the role of osteopontin and its regulation by TRAP in the pathogenesis of common autoimmune disorders.     

Phylogenomic analyses unravel annelid evolution

Annelida, the ringed worms, is a highly diverse animal phylum that includes more than 15,000 described species and constitutes the dominant benthic macrofauna from the intertidal zone down to the deep sea. A robust annelid phylogeny would shape our understanding of animal body-plan evolution and shed light on the bilaterian ground pattern. Traditionally, Annelida has been split into two major groups: Clitellata (earthworms and leeches) and polychaetes (bristle worms), but recent evidence suggests that other taxa that were once considered to be separate phyla (Sipuncula, Echiura and Siboglinidae (also known as Pogonophora)) should be included in Annelida. However, the deep-level evolutionary relationships of Annelida are still poorly understood, and a robust reconstruction of annelid evolutionary history is needed. Here we show that phylogenomic analyses of 34 annelid taxa, using 47,953 amino acid positions, recovered a well-supported phylogeny with strong support for major splits. Our results recover chaetopterids, myzostomids and sipunculids in the basal part of the tree, although the position of Myzostomida remains uncertain owing to its long branch. The remaining taxa are split into two clades: Errantia (which includes the model annelid Platynereis), and Sedentaria (which includes Clitellata). Ancestral character trait reconstructions indicate that these clades show adaptation to either an errant or a sedentary lifestyle, with alteration of accompanying morphological traits such as peristaltic movement, parapodia and sensory perception. Finally, life history characters in Annelida seem to be phylogenetically informative.