RGM is a repulsive guidance molecule for retinal axons

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.     

Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response

Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.     

Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) has an important function in cell regulation both as a precursor of second messenger molecules and by means of its direct interactions with cytosolic and membrane proteins. Biochemical studies have suggested a role for PtdIns(4,5)P2 in clathrin coat dynamics, and defects in its dephosphorylation at the synapse produce an accumulation of coated endocytic intermediates. However, the involvement of PtdIns(4,5)P2 in synaptic vesicle exocytosis remains unclear. Here, we show that decreased levels of PtdIns(4,5)P2 in the brain and an impairment of its depolarization-dependent synthesis in nerve terminals lead to early postnatal lethality and synaptic defects in mice. These include decreased frequency of miniature currents, enhanced synaptic depression, a smaller readily releasable pool of vesicles, delayed endocytosis and slower recycling kinetics. Our results demonstrate a critical role for PtdIns(4,5)P2 synthesis in the regulation of multiple steps of the synaptic vesicle cycle.     

Cytoplasmic PML function in TGF-beta signalling

Transforming growth factor beta (TGF-beta) is a pluripotent cytokine that controls key tumour suppressive functions, but cancer cells are often unresponsive to it. The promyelocytic leukaemia (PML) tumour suppressor of acute promyelocytic leukaemia (APL) accumulates in the PML nuclear body, but cytoplasmic PML isoforms of unknown function have also been described. Here we show that cytoplasmic Pml is an essential modulator of TGF-beta signalling. Pml-null primary cells are resistant to TGF-beta-dependent growth arrest, induction of cellular senescence and apoptosis. These cells also have impaired phosphorylation and nuclear translocation of the TGF-beta signalling proteins Smad2 and Smad3, as well as impaired induction of TGF-beta target genes. Expression of cytoplasmic Pml is induced by TGF-beta. Furthermore, cytoplasmic PML physically interacts with Smad2/3 and SARA (Smad anchor for receptor activation) and is required for association of Smad2/3 with SARA and for the accumulation of SARA and TGF-beta receptor in the early endosome. The PML-RARalpha oncoprotein of APL can antagonize cytoplasmic PML function and APL cells have defects in TGF-beta signalling similar to those observed in Pml-null cells. Our findings identify cytoplasmic PML as a critical TGF-beta regulator, and further implicate deregulated TGF-beta signalling in cancer pathogenesis.     

Critical relative indentation depth in carbon based thinfilms

The thin film hardness estimation by nanoindentation is influenced by substrate beyond a critical relative indentation depth(CRID). In this study we developed a methodology to identify the CRID in amorphous carbon film. Three types of amorphous carbon film deposited on silicon have been studied. The nanoindentation tests were carried out applying a 0.1–10 mN load range on a Berkovich diamond tip, leading to penetration depth-to-film thickness ratios of 8–100%. The work regained during unloading(We) and the work performed during loading(Wt) was estimated for each indentation. The trend of unload-to-load ratio(We/Wt) data as a function of depth has been studied. We/Wtdepth profiles showed a sigmoid trend and the data were fitted by means of a Hill sigmoid equation. Using Hill sigmoid fit and a simple analytical method it is possible to estimate CRID of carbon based films.     

Evolution of genes and genomes on the Drosophila phylogeny

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.     

First result from the Jinping Underground Nuclear Astrophysics experiment JUNA:precise measurement of the 92 keV 25Mg(p,γ)26Al resonance

The 25Mg(p,γ)26Al reaction plays an important role in the study of cosmic 1.809 MeV γ-ray as a signature of ongoing nucleosynthesis in the Galaxy.At astrophysic...     

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

A novel 72-kDa leukocyte-derived osteoglycin enhances the activation of toll-like receptor 4 and exacerbates cardiac inflammation during viral myocarditis

Background

Viral myocarditis can severely damage the myocardium through excessive infiltration of immune cells. Osteoglycin (OGN) is part of the small leucine-rich repeat proteoglycan (SLRP) family. SLRP’s may affect inflammatory and fibrotic processes, but the implication of OGN in cardiac inflammation and the resulting injury upon viral myocarditis is unknown.

Methods and results

This study uncovered a previously unidentified 72-kDa variant of OGN that is predominant in cardiac human and mouse samples of viral myocarditis. Its absence in mice significantly decreased cardiac inflammation and injury in Coxsackievirus-B3-induced myocarditis. It also delayed mortality in lipopolysaccharide-induced endotoxemia going along with a reduced systemic production of pro-inflammatory cytokines. This 72-kDa OGN is expressed in the cell membrane of circulating and resident cardiac macrophages and neutrophils. Co-immunoprecipitation and OGN siRNA experiments revealed that this 72-kDa variant activates the toll-like receptor-4 (TLR4) with a concomitant increase in IL-6, TNF-α, IL-1β, and IL-12 expression. This immune cell activation by OGN occurred via MyD88 and increased phosphorylation of c-jun. Finally, the 72-kDa chondroitin sulfate is the result of O-linked glycosylation of the 32-kDa protein core of OGN. In contrast, the 34-kDa dermatan sulfate-OGN, involved in collagen cross linking, was also the result of O-linked glycosylation.

Conclusion

The current study discovered a novel 72-kDa chondroitin sulfate-OGN that is specific for innate immune cells. This variant is able to bind and activate TLR4. The absence of OGN decreases cytokine production by both circulating and cardiac leukocytes upon (systemic) LPS exposure, and reduces cardiac inflammation and injury in viral myocarditis.