Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA

Set optimization - a rather short introduction

Recent developments in set optimization are surveyed and extended including various set relations as well as fundamental constructions of a convex analysis for set- and vector-valued functions, and duality for set optimization problems. Extensive sections with bibliographical comments summarize the state of the art. Applications to vector optimization and financial risk measures are discussed along with algorithmic approaches to set optimization problems

Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing

<p>Abstract</p> <p>Background</p> <p>Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample.</p> <p>Methods</p> <p>Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing.</p> <p>Results</p> <p>In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples.</p> <p>Conclusion</p> <p>MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.</p

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

OXPHOS Supercomplexes as a Hallmark of the Mitochondrial Phenotype of Adipogenic Differentiated Human MSCs

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation

The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Chondroitin sulfate proteoglycans are major inhibitory molecules for neural plasticity under both physiological and pathological conditions. The chondroitin sulfate degrading enzyme chondroitinase ABC promotes functional recovery after spinal cord injury, and restores experience-dependent plasticity, such as ocular dominance plasticity and fear erasure plasticity, in adult rodents. These data suggest that the sugar chain in a proteoglycan moiety is essential for the inhibitory activity of proteoglycans. However, the significance of the core protein has not been studied extensively. Furthermore, considering that chondroitinase ABC is derived from bacteria, a mammalian endogenous enzyme which can inactivate the proteoglycans' activity is desirable for clinical use.</p> <p>Methods</p> <p>The degradation activity of ADAMTS-4 was estimated for the core proteins of chondroitin sulfate proteoglycans, that is, brevican, neurocan and phosphacan. To evaluate the biological significance of ADMATS-4 activity, an <it>in vitro </it>neurite growth assay and an <it>in vivo </it>neuronal injury model, spinal cord contusion injury, were employed.</p> <p>Results</p> <p>ADAMTS-4 digested proteoglycans, and reversed their inhibition of neurite outgrowth. Local administration of ADAMTS-4 significantly promoted motor function recovery after spinal cord injury. Supporting these findings, the ADAMTS-4-treated spinal cord exhibited enhanced axonal regeneration/sprouting after spinal cord injury.</p> <p>Conclusions</p> <p>Our data suggest that the core protein in a proteoglycan moiety is also important for the inhibition of neural plasticity, and provides a potentially safer tool for the treatment of neuronal injuries.</p

Angiogenesis is associated with the onset of hyperplasia in human ductal breast disease

BACKGROUND: The precise timing of the angiogenic switch and the role of angiogenesis in the development of breast malignancy is currently unknown. METHODS: Therefore, the expression of CD31 (pan endothelial cells (ECs)), endoglin (actively proliferating ECs), hypoxia-inducible factor-1 (HIF-1alpha), vascular endothelial growth factor-A (VEGF) and tissue factor (TF) were quantified in 140 surgical specimens comprising normal human breast, benign and pre-malignant hyperplastic tissue, in situ and invasive breast cancer specimens. RESULTS: Significant increases in angiogenesis (microvessel density) were observed between normal and benign hyperplastic breast tissue (P<0.005), and between in situ and invasive carcinomas (P<0.0005). In addition, significant increases in proliferating ECs were observed in benign hyperplastic breast compared with normal breast (P<0.05) cancers and in invasive compared with in situ cancers (P<0.005). Hypoxia-inducible factor-1alpha, VEGF and TF expression were significantly associated with increases in both angiogenesis and proliferating ECs (P<0.05). Moreover, HIF-1alpha was expressed by 60-75% of the hyperplastic lesions, and a significant association was observed between VEGF and TF in ECs (P<0.005) and invasive tumour cells (P<0.01). CONCLUSIONS: These findings are the first to suggest that the angiogenic switch, associated with increases in HIF-1alpha, VEGF and TF expression, occurs at the onset of hyperplasia in the mammary duct, although the greatest increase in angiogenesis occurs with the development of invasion

New Insights into the Mechanisms of Embryonic Stem Cell Self-Renewal under Hypoxia: A Multifactorial Analysis Approach

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and non-physiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O2 tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection

Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction