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Find video protocols related to scientific articles indexed in Pubmed.
Malate synthesis and secretion mediated by a Mn enhanced malate dehydrogenase, SgMDH1, confers superior Mn tolerance in Stylosanthes guianensis.
Plant Physiol.
PUBLISHED: 11-08-2014
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Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in stylo (Stylosanthes guianensis) has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in stylo was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two stylo genotypes, which revealed that Fine-stem had higher Mn tolerance than TPRC2001-1, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn stimulated increases in malate concentrations and exudation rate were only observed in Fine-stem. Proteomic analysis of Fine-stem roots revealed a malate dehydrogenase, SgMDH1 accumulated in response to Mn toxicity. Western blot and quantitative PCR analysis showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1 mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, and in vivo increased malate concentrations in yeast, soybean (Glycine max) hairy roots and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of stylo is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels.
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IKK? is an IRF5 kinase that instigates inflammation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-19-2014
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The transcription factor interferon regulatory factor 5 (IRF5) is essential for the induction of inflammatory cytokines, but the mechanism by which IRF5 is activated is not well understood. Here we present evidence that the kinase IKK? phosphorylates and activates IRF5 in response to stimulation in several inflammatory pathways, including those emanated from Toll-like receptors and retinoic acid-inducible gene I-like receptors. IKK? phosphorylates mouse IRF5 at specific residues, including serine 445 (S446 in human IRF5 isoform 1), as evidenced by mass spectrometry analysis and detection with a phosphospecific antibody. Recombinant IKK? phosphorylated IRF5 at Ser-445 in vitro, and a point mutation of this serine abolished IRF5 activation and cytokine production. Depletion or pharmacologic inhibition of IKK? prevented IRF5 phosphorylation. These results indicate that IKK? is an IRF5 kinase that instigates inflammation.
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Association of PCB, PBDE and PCDD/F body burdens with hormone levels for children in an e-waste dismantling area of Zhejiang Province, China.
Sci. Total Environ.
PUBLISHED: 08-29-2014
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Increased electronic waste (e-waste) has raised public concerns regarding exposure to numerous toxic contaminants, particularly polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). In China, the body burdens of PCBs, PBDEs and PCDD/Fs are associated with thyroid hormones in populations from e-waste dismantling sites; however, it is unclear whether this association occurs in children. In this study, we determined the serum levels of PCBs, PBDEs and PCDD/Fs and the endocrine hormones including free triiodothyronine (FT3), total triiodothyronine (TT3), free thyroxine (FT4), total thyroxine (TT4), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), cortisol and growth hormone (GH) in 21 children from an e-waste dismantling area and 24 children from a control area. The results showed that the mean levels of ?PCBs and ?PBDEs in the exposure group were significantly higher than in the control group (40.56 and 32.09 ng g(-1) lipid vs. 20.69 and 8.43 ng g(-1) lipid, respectively, p<0.01 for each), and the mean level of ?PCDD/Fs in the exposure group was higher than in the control group, but the difference was not significant (206.17 vs. 160.27 pg g(-1) lipid, p>0.05). For the endocrine hormones, we did not find significant differences between the exposed and control groups, although the mean levels of FT3, TT3, TT4, ACTH, cortisol and GH were higher, whereas the mean levels of FT4 and TSH were lower in the exposed group. The mean level of ?PBDEs was positively correlated with the mean levels of ?PCBs (r=0.60, p<0.05) and ?PCDD/Fs (r=0.61, p<0.05). Furthermore, the mean level of ?PBDEs was positively correlated with ACTH (r=0.61, p<0.05). In conclusion, our data suggested that exposure to e-waste dismantling environment increased the body burdens of PCBs and PBDEs in local children and that these contaminants released from the e-waste might contribute to abnormal changes in hormone levels.
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A hydrophobic hole transporting oligothiophene for planar perovskite solar cells with improved stability.
Chem. Commun. (Camb.)
PUBLISHED: 08-12-2014
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An oligothiophene derivative named DR3TBDTT with high hydrophobicity was synthesized and functioned as the hole transporting material without an ion additive. 8.8% of power conversion efficiency was obtained for CH3NH3PbI3-xClx based planar solar cells with improved stability, compared to devices using Li-TFSI doped spiro-MeOTAD.
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A note on the efficiencies of sampling strategies in two-stage bayesian regional fine mapping of a quantitative trait.
Genet. Epidemiol.
PUBLISHED: 08-01-2014
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In focused studies designed to follow up associations detected in a genome-wide association study (GWAS), investigators can proceed to fine-map a genomic region by targeted sequencing or dense genotyping of all variants in the region, aiming to identify a functional sequence variant. For the analysis of a quantitative trait, we consider a Bayesian approach to fine-mapping study design that incorporates stratification according to a promising GWAS tag SNP in the same region. Improved cost-efficiency can be achieved when the fine-mapping phase incorporates a two-stage design, with identification of a smaller set of more promising variants in a subsample taken in stage 1, followed by their evaluation in an independent stage 2 subsample. To avoid the potential negative impact of genetic model misspecification on inference we incorporate genetic model selection based on posterior probabilities for each competing model. Our simulation study shows that, compared to simple random sampling that ignores genetic information from GWAS, tag-SNP-based stratified sample allocation methods reduce the number of variants continuing to stage 2 and are more likely to promote the functional sequence variant into confirmation studies.
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Improved light absorption and charge transport for perovskite solar cells with rough interfaces by sequential deposition.
Nanoscale
PUBLISHED: 06-14-2014
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Recently, highly efficient solar cells based on organic-inorganic perovskites have been intensively reported for developing fabricating methods and device structures. Additional power conversion efficiency should be gained without increasing the thickness and the complexity of the devices to accord with practical applications. In this paper, a rough interface between perovskite and HTM was fabricated in perovskite solar cells to enhance the light scattering effect and improve the charge transport. The parameters related to the morphology have been systematically investigated by sequential deposition. Simultaneous enhancements of short-circuit current and power conversion efficiency were observed in both CH?NH?PbI? and CH?NH?PbI?-xClx devices containing the rough interface, with power conversion efficiencies of 10.2% and 10.8%, respectively. Our finding provides an efficient and universal way to control the morphology and further optimize perovskite solar cells for devices by sequential deposition with various structures.
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Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-27-2014
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Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(?TrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
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A highly efficient mesoscopic solar cell based on CH?NH?PbI(3-x)Cl(x) fabricated via sequential solution deposition.
Chem. Commun. (Camb.)
PUBLISHED: 05-24-2014
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A mixed halide perovskite of CH3NH3PbI(3-x)Cl(x) is synthesized via two-step sequential solution deposition by using a mixture of PbCl2 and PbI2 as the precursor to overcome the low solubility of pure PbCl2 with easy morphology control. 11.7% power conversion efficiency is achieved for the mesoscopic cell, much higher than the cell constructed via a spin-coating process.
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Vertical phase separation in bulk heterojunction solar cells formed by in situ polymerization of fulleride.
Sci Rep
PUBLISHED: 05-01-2014
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Vertical phase separation of the donor and the acceptor in organic bulk heterojunction solar cells is crucial to improve the exciton dissociation and charge transport efficiencies. This is because whilst the exciton diffusion length is limited, the organic film must be thick enough to absorb sufficient light. However, it is still a challenge to control the phase separation of a binary blend in a bulk heterojunction device architecture. Here we report the realization of vertical phase separation induced by in situ photo-polymerization of the acrylate-based fulleride. The power conversion efficiency of the devices with vertical phase separation increased by 20%. By optimising the device architecture, the power conversion efficiency of the single junction device reached 8.47%. We believe that in situ photo-polymerization of acrylate-based fulleride is a universal and controllable way to realise vertical phase separation in organic blends.
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The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling.
Mol. Cell
PUBLISHED: 04-29-2014
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The innate immune system deploys a variety of sensors to detect signs of infection. Nucleic acids represent a major class of pathogen signatures that can trigger robust immune responses. The presence of DNA in the cytoplasm of mammalian cells is a danger signal that activates innate immune responses; however, how cytosolic DNA triggers these responses remained unclear until recently. In this review, we focus on the mechanism of DNA sensing by the newly discovered cGAS-cGAMP-STING pathway and highlight recent progress in dissecting the in vivo functions of this pathway in immune defense as well as autoimmunity.
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Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway.
PLoS Pathog.
PUBLISHED: 04-01-2014
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Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.
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Innate immune sensing and signaling of cytosolic nucleic acids.
Annu. Rev. Immunol.
PUBLISHED: 03-25-2014
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The innate immune system utilizes pattern-recognition receptors (PRRs) to detect the invasion of pathogens and initiate host antimicrobial responses such as the production of type I interferons and proinflammatory cytokines. Nucleic acids, which are essential genetic information carriers for all living organisms including viral, bacterial, and eukaryotic pathogens, are major structures detected by the innate immune system. However, inappropriate detection of self nucleic acids can result in autoimmune diseases. PRRs that recognize nucleic acids in cells include several endosomal members of the Toll-like receptor family and several cytosolic sensors for DNA and RNA. Here, we review the recent advances in understanding the mechanism of nucleic acid sensing and signaling in the cytosol of mammalian cells as well as the emerging role of cytosolic nucleic acids in autoimmunity.
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Structural basis for the prion-like MAVS filaments in antiviral innate immunity.
Elife
PUBLISHED: 02-27-2014
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Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an ?-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.
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Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.
Nature
PUBLISHED: 02-11-2014
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Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.
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A catalytic-independent role for the LUBAC in NF-?B activation upon antigen receptor engagement and in lymphoma cells.
Blood
PUBLISHED: 02-04-2014
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Antigen receptor-mediated nuclear factor ?B (NF-?B) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-?B activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-?B activation, dynamically integrates the CBM and marshals NF-?B optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-?B activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.
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K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking.
Mol. Cell
PUBLISHED: 01-28-2014
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Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.
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A novel mitochondrial MAVS/Caspase-8 platform links RNA virus-induced innate antiviral signaling to Bax/Bak-independent apoptosis.
J. Immunol.
PUBLISHED: 01-03-2014
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Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2- or Bcl-xL-overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-? but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.
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Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation.
Cell
PUBLISHED: 01-02-2014
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Pathogens and cellular danger signals activate sensors such as RIG-I and NLRP3 to produce robust immune and inflammatory responses through respective adaptor proteins MAVS and ASC, which harbor essential N-terminal CARD and PYRIN domains, respectively. Here, we show that CARD and PYRIN function as bona fide prions in yeast and that their prion forms are inducible by their respective upstream activators. Likewise, a yeast prion domain can functionally replace CARD and PYRIN in mammalian cell signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, a conserved fungal NOD-like receptor and prion pair can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion-like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.
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The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop.
Cell Rep
PUBLISHED: 01-02-2014
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The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.
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microRNA-21 promotes cardiac fibrosis and development of heart failure with preserved left ventricular ejection fraction by up-regulating Bcl-2.
Int J Clin Exp Pathol
PUBLISHED: 01-01-2014
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The morbidity and mortality of heart failure with preserved left ventricular ejection fraction (HFpEF) were similar to those of systolic heart failure, but the pathogenesis of HFpEF remains poorly understood. It was demonstrated that, in systolic heart failure, microRNA-21 (miR-21) could inhibit the apoptosis of cardiac fibroblasts, leading to cardiac hypertrophy and myocardial fibrosis, but the role of miR-21 in HFpEF remains unknown. By employing cell culture technique, rat myocardiocytes and cardiac fibroblasts were obtained. The expression of miR-21 in the two cell types under different conditions was compared and we found that the miR-21 expression was significantly higher in cardiac fibroblasts than in myocardiocytes. We established a rat HFpEF model and harvested the tissues of cardiac apex for pathological examination, Northern blotting and so forth. We found that miR-21 expression was significantly higher in model rats than in sham-operated rats, and the model rats developed the cardiac atrophy and cardiac fibrosis. After injection of miR-21 antagonist, the the cardiac atrophy and cardiac fibrosis were conspicuously ameliorated. Both in vivo and in vitro, inhibition of miR-21 expression resulted in reduced Bcl-2 expression while over-expression of miR-21 led to elevation of Bcl-2 expression. Our study suggested that miR-21 promoted the development of HFpEF by up-regulating the expression of anti-apoptotic gene Bcl-2 and thereby suppressing the apoptosis of cardiac fibrosis.
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Superior aluminium (Al) tolerance of Stylosanthes is achieved mainly by malate synthesis through an Al-enhanced malic enzyme, SgME1.
New Phytol.
PUBLISHED: 08-31-2013
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Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism. A novel malic enzyme, SgME1, was identified from the Al-tolerant genotype TPRC2001-1 after 72 h Al exposure by two-dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean (Phaseolus vulgaris) hairy roots. Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001-1, as indicated by 5.8-fold higher root malate concentrations and approximately two-fold higher Al concentrations in roots and root symplasts of TPRC2001-1 than those of the Al-sensitive genotype Fine-stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001-1. Gene and protein expression of SgME1 was only enhanced in TPRC2001-1 after 72 h Al exposure. Overexpressing SgME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation. These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al-enhanced malate synthesis, functionally controlled by SgME1.
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Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects.
Science
PUBLISHED: 08-29-2013
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Invasion of microbial DNA into the cytoplasm of animal cells triggers a cascade of host immune reactions that help clear the infection; however, self DNA in the cytoplasm can cause autoimmune diseases. Biochemical approaches led to the identification of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here, we show that cells from cGAS-deficient (cGas(-/-)) mice, including fibroblasts, macrophages, and dendritic cells, failed to produce type I interferons and other cytokines in response to DNA transfection or DNA virus infection. cGas(-/-) mice were more susceptible to lethal infection with herpes simplex virus 1 (HSV1) than wild-type mice. We also show that cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice.
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Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.
Science
PUBLISHED: 08-08-2013
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Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-? induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.
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Marginal analysis of longitudinal ordinal data with misclassification in both response and covariates.
Biom J
PUBLISHED: 05-15-2013
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Marginal methods have been widely used for the analysis of longitudinal ordinal and categorical data. These models do not require full parametric assumptions on the joint distribution of repeated response measurements but only specify the marginal or even association structures. However, inference results obtained from these methods often incur serious bias when variables are subject to error. In this paper, we tackle the problem that misclassification exists in both response and categorical covariate variables. We develop a marginal method for misclassification adjustment, which utilizes second-order estimating functions and a functional modeling approach, and can yield consistent estimates and valid inference for mean and association parameters. We propose a two-stage estimation approach for cases in which validation data are available. Our simulation studies show good performance of the proposed method under a variety of settings. Although the proposed method is phrased to data with a longitudinal design, it also applies to correlated data arising from clustered and family studies, in which association parameters may be of scientific interest. The proposed method is applied to analyze a dataset from the Framingham Heart Study as an illustration.
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Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.
Mol. Cell
PUBLISHED: 05-08-2013
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The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2-OH of GMP and 5-phosphate of AMP, and the other between 3-OH of AMP and 5-phosphate of GMP. This molecule, termed 23-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 23-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.
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A prognostic model for distant metastasis in locally advanced nasopharyngeal carcinoma after concurrent chemoradiotherapy.
Head Neck
PUBLISHED: 04-24-2013
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Background: A prognostic model should be established for distant metastasis in locally advanced nasopharyngeal carcinoma (LA-NPC) after concurrent chemoradiotherapy (CCRT). Methods: LA-NPC patients received CCRT were divided into a construction set (230 patients) and a validating set (115 patients). The constructed index was derived on the former, then tested on the latter. Results: The prognostic score was de?ned as the number of adverse prognostic factors: age>45, N3 category, Hemoglobin<11.0g/dL and Lactate dehydrogenase?240U/L. The score predicted the 5y-distant metastasis-free survival as follow: 0, 91%; 1, 74%; 2, 51%; ?3, 12%. In the validating set, the observed 5y-distant metastasis-free survival of these four groups with score 0, 1, 2 and 3 or higher were 81%, 68%, 47% and 15%, respectively. Conclusion: The established model might be useful for predicting the risk of distant metastasis in LA-NPC patients underwent concurrent chemoradiotherapy and may identify patients need intensified adjuvant chemotherapy. Head Neck, 2013.
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Are there age-related differences in social suggestibility to central and peripheral misinformation?
Exp Aging Res
PUBLISHED: 04-24-2013
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BACKGROUND/STUDY CONTEXT: Dalton and Daneman ( 2006 , Memory, 14, 486-501) showed that young adults can be induced to accept misinformation from a co-witness, even if it contradicts central features of a previously witnessed event. This study investigated whether older adults are also susceptible to social suggestion, and if so, whether to the same or different degree as their younger counterparts. The study also investigated whether participants were more likely to succumb to suggestions delivered by a peer or an older figure.
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Advanced nasopharyngeal carcinoma radiotherapy with volumetric modulated arcs and the potential role of flattening filter-free beams.
Radiat Oncol
PUBLISHED: 04-14-2013
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The purpose of this study is to investigate the dosimetric characteristics of volumetric modulated arc therapy (VMAT) with flattening filter-free (FFF) beams and assess the role of VMAT in the treatment of advanced nasopharyngeal carcinoma (NPC).
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Working memory inefficiency: minimal information is utilized in visual recognition tasks.
J Exp Psychol Learn Mem Cogn
PUBLISHED: 02-18-2013
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Can people make perfect use of task-relevant information in working memory (WM)? Specifically, when questioned about an item in an array that does not happen to be in WM, can participants take into account other items that are in WM, eliminating them as response candidates? To address this question, an ideal-responder model that assumes perfect use of items in a capacity-limited WM was tested against a minimal-responder model that assumes use of only information about the queried item. Three different WM tasks were adopted: change detection, identity recognition, and location recognition. The change-detection task produced benchmark WM results. The 2 novel tasks showed that only the minimal responder model provided convergence with this benchmark. This finding was replicable even when the change-detection task was replaced by a feature-switch detection task. Thus, it appears that people do not make full use of information in WM.
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Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor.
J. Lipid Res.
PUBLISHED: 02-18-2013
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Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells.
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RNA helicase signaling is critical for type i interferon production and protection against Rift Valley fever virus during mucosal challenge.
J. Virol.
PUBLISHED: 02-13-2013
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Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
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Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-04-2013
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In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P<0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P<0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P<0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.
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Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.
Cell
PUBLISHED: 01-30-2013
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Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.
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XRCC1 codon 399Gln polymorphism is associated with radiotherapy-induced acute dermatitis and mucositis in nasopharyngeal carcinoma patients.
Radiat Oncol
PUBLISHED: 01-29-2013
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To evaluate the association between single nucleotide polymorphisms (SNPs) at the 194 and 399 codons of XRCC1, and the risk of severe acute skin and oral mucosa reactions in nasopharyngeal carcinoma patients in China.
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Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination.
Cell
PUBLISHED: 01-24-2013
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Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
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IKK?-mediated tumorigenesis requires K63-linked polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex.
Cell Rep
PUBLISHED: 01-22-2013
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I?B kinase ? (IKK?, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ~30% of breast cancers, that promotes malignant transformation through NF-?B activation. Here, we show that IKK? is modified and regulated by K63-linked polyubiquitination at lysine 30 and lysine 401. Tumor necrosis factor alpha and interleukin-1? stimulation induces IKK? K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKK? kinase activity, IKK?-mediated NF-?B activation, and IKK?-induced malignant transformation. Disruption of K63-linked ubiquitination of IKK? does not affect its overall structure but impairs the recruitment of canonical NF-?B proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKK?. Altogether, these observations demonstrate that K63-linked polyubiquitination regulates IKK? activity in both inflammatory and oncogenic contexts and suggests an alternative approach to targeting this breast cancer oncogene.
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Regulation of NF-?B by ubiquitination.
Curr. Opin. Immunol.
PUBLISHED: 01-08-2013
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The nuclear factor ? enhancer binding protein (NF-?B) family of transcription factors regulates the expression of a large array of genes involved in diverse cellular processes including inflammation, immunity and cell survival. Activation of NF-?B requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Studies in the past few years have provided new insights into the mechanisms underlying regulation of NF-?B by ubiquitination, including the involvement of multiple linkages of ubiquitin, the essential role of ubiquitin binding, and the function of unanchored polyubiquitin chains. In this review, we will focus on recent advances in understanding the role of ubiquitination in NF-?B regulation in various pathways.
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MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades.
Elife
PUBLISHED: 01-01-2013
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RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-?B and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-?B activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI:http://dx.doi.org/10.7554/eLife.00785.001.
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The role of ubiquitylation in immune defence and pathogen evasion.
Nat. Rev. Immunol.
PUBLISHED: 12-09-2011
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Ubiquitylation is a widely used post-translational protein modification that regulates many biological processes, including immune responses. The role of ubiquitin in immune regulation was originally uncovered through studies of antigen presentation and the nuclear factor-?B family of transcription factors, which orchestrate host defence against microorganisms. Recent studies have revealed crucial roles of ubiquitylation in many aspects of the immune system, including innate and adaptive immunity and antimicrobial autophagy. In addition, mounting evidence indicates that microbial pathogens exploit the ubiquitin pathway to evade the host immune system. Here, we review recent advances on the role of ubiquitylation in host defence and pathogen evasion.
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Persistent stimulation with interleukin-17 desensitizes cells through SCF?-TrCP-mediated degradation of Act1.
Sci Signal
PUBLISHED: 11-03-2011
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The proinflammatory cytokine interleukin-17 (IL-17) is important for the immune response to pathogens and also contributes to the pathogenesis of various inflammatory diseases. To avoid persistent inflammation, signaling by the IL-17 receptor (IL-17R), which involves the adaptor protein Act1, must be tightly controlled. Here, we report that persistent stimulation of HeLa cells with IL-17 resulted in degradation of Act1 and desensitization of IL-17R signaling. IL-17 stimulated the Lys48-linked polyubiquitination and degradation of Act1, which was phosphorylation-dependent, similar to the IL-17-dependent degradation of inhibitor of nuclear factor ?B ?. Act1 was recruited to SCF (Skp1-cullin-1-F-box)-type E3 ubiquitin ligase complexes containing ?-transducin repeat-containing protein 1 (?-TrCP1) or ?-TrCP2 in a phosphorylation-dependent manner upon stimulation of cells with IL-17. Dominant-negative ?-TrCP or knockdown of ?-TrCP1 and ?-TrCP2 markedly reduced IL-17-induced, phosphorylation-dependent ubiquitination and degradation of Act1. Thus, our studies identify a previously uncharacterized desensitization mechanism, involving the SCF?-TrCP-mediated degradation of Act1, that occurs during persistent stimulation with IL-17.
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Mitochondrial antiviral signaling protein (MAVS) monitors commensal bacteria and induces an immune response that prevents experimental colitis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-29-2011
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RIG-I-like receptors (RLRs) activate host innate immune responses against virus infection through recruiting the mitochondrial adaptor protein MAVS (also known as IPS1, VISA, or CARDIF). Here we show that MAVS also plays a pivotal role in maintaining intestinal homeostasis. We found that MAVS knockout mice developed more severe mortality and morbidity than WT animals in an experimental model of colitis. Bone marrow transplantation experiments revealed that MAVS in cells of nonhematopoietic origin plays a dominant role in the protection against colitis. Importantly, RNA species derived from intestinal commensal bacteria activate the RIG-I-MAVS pathway to induce the production of multiple cytokines and antimicrobial peptides, including IFN-? and RegIII?. These results unveil a previously unexplored role of MAVS in monitoring intestinal commensal bacteria and maintaining tissue homeostasis.
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HSV infection induces production of ROS, which potentiate signaling from pattern recognition receptors: role for S-glutathionylation of TRAF3 and 6.
PLoS Pathog.
PUBLISHED: 07-18-2011
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The innate immune response constitutes the first line of defense against infections. Pattern recognition receptors recognize pathogen structures and trigger intracellular signaling pathways leading to cytokine and chemokine expression. Reactive oxygen species (ROS) are emerging as an important regulator of some of these pathways. ROS directly interact with signaling components or induce other post-translational modifications such as S-glutathionylation, thereby altering target function. Applying live microscopy, we have demonstrated that herpes simplex virus (HSV) infection induces early production of ROS that are required for the activation of NF-?B and IRF-3 pathways and the production of type I IFNs and ISGs. All the known receptors involved in the recognition of HSV were shown to be dependent on the cellular redox levels for successful signaling. In addition, we provide biochemical evidence suggesting S-glutathionylation of TRAF family proteins to be important. In particular, by performing mutational studies we show that S-glutathionylation of a conserved cysteine residue of TRAF3 and TRAF6 is important for ROS-dependent activation of innate immune pathways. In conclusion, these findings demonstrate that ROS are essential for effective activation of signaling pathways leading to a successful innate immune response against HSV infection.
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Enhancement of the power conversion efficiency by expanding the absorption spectrum with fluorescence layers.
Opt Express
PUBLISHED: 06-07-2011
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The spectral response of Poly(3-hexylthiophene) (P3HT): 1-(3-methoxycarbonyl)-propyl-1-phenyl-(6,6)C61 (PCBM) heterojunction film is between 350 nm and 650 nm, meaning that a lot of the sunlight is lost at ultraviolet and infrared regions. We fabricated solar cells by the attachment of a fluorescence layer which absorbs UV light, and emit visible light which will be re-used by P3HT, and thus the absorption spectrum is expanded. Since N,N-bis(3-methylphenyl)-N,N-bis(phenyl)-benzidine (TPD) has high reflectance in the visible range, the usage of UV light will not manifest; when LiF is added as an antireflection layer, PCE was enhanced from 2.50% to 2.68%.
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Identification of differentially expressed proteins in soybean nodules under phosphorus deficiency through proteomic analysis.
Proteomics
PUBLISHED: 05-02-2011
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Symbiotic nitrogen fixation is a high-phosphorus demand process. Proteomic analysis was performed to identify the differentially expressed proteins in soybean nodules under phosphate starvation, and qRT-PCR was subsequently conducted to examine the expression patterns of the genes encoding the identified proteins. There were 44 phosphate-starvation responsive proteins identified from soybean nodules. Among them, 14 plant and 3 rhizobial proteins were up-regulated, whereas 13 plant and 14 rhizobial proteins were down-regulated by phosphate starvation. The qRT-PCR assays verified that gene expression correlated with 11 of the 14 up-regulated proteins from plants, but only 4 of 13 down-regulated proteins were correlated to the expression of the corresponding genes, suggesting that most up-regulated proteins may be controlled at the transcriptional level, whereas down-regulated proteins were controlled at the post-transcriptional level. Furthermore, a group of genes exhibited differential responses to phosphate starvation in nodules versus roots, suggesting that different adaptive responses might occur between roots and nodules. To our best knowledge, this is the first study to reveal differential protein profiles of nodules responding to phosphate starvation through proteomic analysis, which could result in a relatively comprehensive understanding of molecular mechanisms through which soybean nodules adapt to phosphorus stress.
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Viperin links lipid bodies to immune defense.
Immunity
PUBLISHED: 03-26-2011
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Viperin is an interferon-stimulated gene that exerts antiviral effects. In this issue of Immunity, Saitoh et al. (2011) uncovered an unexpected function of Viperin and lipid bodies in interferon induction by Toll-like receptors, specifically in plasmacytoid dendritic cells.
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Direct, noncatalytic mechanism of IKK inhibition by A20.
Mol. Cell
PUBLISHED: 02-14-2011
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A20 is a potent anti-inflammatory protein that inhibits NF-?B, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the I?B kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20s seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.
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MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.
Cell
PUBLISHED: 02-08-2011
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In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-?B to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.
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NLRX1 negatively regulates TLR-induced NF-?B signaling by targeting TRAF6 and IKK.
Immunity
PUBLISHED: 01-31-2011
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Tight regulation of NF-?B signaling is essential for innate and adaptive immune responses, yet the molecular mechanisms responsible for its negative regulation are not completely understood. Here, we report that NLRX1, a NOD-like receptor family member, negatively regulates Toll-like receptor-mediated NF-?B activation. NLRX1 interacts with TRAF6 or I?B kinase (IKK) in an activation signal-dependent fashion. Upon LPS stimulation, NLRX1 is rapidly ubiquitinated, disassociates from TRAF6, and then binds to the IKK complex, resulting in inhibition of IKK? and IKK? phosphorylation and NF-?B activation. Knockdown of NLRX1 in various cell types markedly enhances IKK phosphorylation and the production of NF-?B-responsive cytokines after LPS stimulation. We further provide in vivo evidence that NLRX1 knockdown in mice markedly enhances susceptibility to LPS-induced septic shock and plasma IL-6 level. Our study identifies a previously unrecognized role for NLRX1 in the negative regulation of TLR-induced NF-?B activation by dynamically interacting with TRAF6 and the IKK complex.
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Cc2d1a, a C2 domain containing protein linked to nonsyndromic mental retardation, controls functional maturation of central synapses.
J. Neurophysiol.
PUBLISHED: 01-27-2011
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Cc2d1a is an evolutionarily conserved protein composed of NH(2)-terminal Drosophila melanogaster 14 domain (DM14) domains and a COOH-terminal C2 domain. Human patients with homozygotic mutation in the gene suffer from nonsyndromic mental retardation, implying that Cc2d1a functions in the central nervous system. To examine the physiological role of the Cc2d1a, we generated and analyzed Cc2d1a knockout (KO) mice. Cc2d1a KO mice die soon after birth, apparently because of their inability to breathe. Histological analysis of Cc2d1a KO animals did not identify any structural defects in the peripheral respiratory apparatus. However, functional analysis of synapses formed between Cc2d1a-deficient cortical neurons revealed a robust increase in the pace of maturation of evoked synaptic responses as well as synaptic vesicle trafficking. This synaptic anomaly was rescued by reintroducing full-length Cc2d1a but not C2-domain-deletion mutant, underscoring the functional importance of C2 domain. Our data suggest that Cc2d1a is required for mouse survival and performs essential function in controlling functional maturation of synapses.
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Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling.
Dev. Cell
PUBLISHED: 01-06-2011
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Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular "tubulogenesis" are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.
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Expanding role of ubiquitination in NF-?B signaling.
Cell Res.
PUBLISHED: 12-07-2010
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Best known for its role in targeting protein degradation by the proteasome, ubiquitin modification has also emerged as an important mechanism that regulates cell signaling through proteasome-independent mechanisms. The role of ubiquitin as a versatile signaling tag is characteristically illustrated in the NF-?B pathways, which regulate a variety of physiological and pathological processes in response to diverse stimuli. Here, we review the role of ubiquitination in different steps of the NF-?B signaling cascades, focusing on recent advances in understanding the mechanisms of protein kinase activation by polyubiquitin chains in different pathways that converge on NF-?B.
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High-performance single CdS nanowire (nanobelt) Schottky junction solar cells with Au/graphene Schottky electrodes.
ACS Appl Mater Interfaces
PUBLISHED: 11-08-2010
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High-performance single CdS nanowire (NW) as well as nanobelt (NB) Schottky junction solar cells were fabricated. Au (5 nm)/graphene combined layers were used as the Schottky contact electrodes to the NWs (NBs). Typical as-fabricated NW solar cell shows excellent photovoltaic behavior with an open circuit voltage of ?0.15 V, a short circuit current of ?275.0 pA, and an energy conversion efficiency of up to ?1.65%. The physical mechanism of the combined Schottky electrode was discussed. We attribute the prominent capability of the devices to the high-performance Schottky combined electrode, which has the merits of low series resistance, high transparency, and good Schottky contact to the CdS NW (NB). Besides, a promising site-controllable patterned graphene transfer method, which has the advantages of economizing graphene material and free from additional etching process, was demonstrated in this work. Our results suggest that semiconductor NWs (NBs) are promising materials for novel solar cells, which have potential application in integrated nano-optoelectronic systems.
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Arrhythmogenic autoantibodies against calcium channel lead to sudden death in idiopathic dilated cardiomyopathy.
Eur. J. Heart Fail.
PUBLISHED: 11-02-2010
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Calcium channel plays an important role in the autoimmune pathogenesis of idiopathic dilated cardiomyopathy (DCM). Autoantibodies have emerged as a new upstream target of sudden death in DCM. We sought to validate the hypothesis that autoantibodies against l-type calcium channel (CC-AAbs) are arrhythmogenic and lead to sudden death in patients with DCM.
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Recent progresses on materials for electrophosphorescent organic light-emitting devices.
Adv. Mater. Weinheim
PUBLISHED: 08-29-2010
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Although organic light-emitting devices have been commercialized as flat panel displays since 1997, only singlet excitons were emitted. Full use of singlet and triplet excitons, electrophosphorescence, has attracted increasing attentions after the premier work made by Forrest, Thompson, and co-workers. In fact, red electrophosphorescent dye has already been used in sub-display of commercial mobile phones since 2003. Highly efficient green phosphorescent dye is now undergoing of commercialization. Very recently, blue phosphorescence approaching the theoretical efficiency has also been achieved, which may overcome the final obstacle against the commercialization of full color display and white light sources from phosphorescent materials. Combining light out-coupling structures with highly efficient phosphors (shown in the table-of-contents image), white emission with an efficiency matching that of fluorescent tubes (90 lm/W) has now been realized. It is possible to tune the color to the true white region by changing to a deep blue emitter and corresponding wide gap host and transporting material for the blue phosphor. In this article, recent progresses in red, green, blue, and white electrophosphorescent materials for OLEDs are reviewed, with special emphasis on blue electrophosphorescent materials.
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Emerging role of ISG15 in antiviral immunity.
Cell
PUBLISHED: 08-02-2010
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Cells express a plethora of interferon-stimulated genes (ISGs) in response to viral infection. Among these is ISG15, a ubiquitin-like protein (UBL) that can be covalently attached to both host and viral proteins. Here we review recent advances toward understanding the role and mechanism of ISG15 modification in antiviral defense.
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Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.
PLoS Pathog.
PUBLISHED: 06-16-2010
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Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68), a model herpesvirus for human Kaposis sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA). Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.
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CC2D1A, a DM14 and C2 domain protein, activates NF-kappaB through the canonical pathway.
J. Biol. Chem.
PUBLISHED: 06-07-2010
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CC2D1A is an evolutionarily conserved protein that contains four DM14 domains at the N terminus and a C2 domain at the C terminus. Loss-of-function mutations in CC2D1A have been linked to mental retardation in human, but the biochemical function of this protein is largely unknown. Here, we show that CC2D1A is a potent activator of NF-kappaB. The activation of NF-kappaB by CC2D1A requires its C2 domain. CC2D1A activates NF-kappaB in a manner that depends on the ubiquitin-conjugating enzyme Ubc13, TNF receptor-associated factor TRAF2, the protein kinase TAK1, and the IkappaB kinase (IKK) complex. In addition, the deubiquitination enzyme Cylindromatosis (CYLD) negatively regulates the activity of CC2D1A. These results suggest that CC2D1A activates NF-kappaB through the canonical IKK pathway.
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A novel missense mutation in the nuclear factor-?B essential modulator (NEMO) gene resulting in impaired activation of the NF-?B pathway and a unique clinical phenotype presenting as MRSA subdural empyema.
J. Clin. Immunol.
PUBLISHED: 04-23-2010
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We describe a previously unreported 437 T?G missense mutation producing a V146G substitution in the first coiled-coil (CC1) domain of nuclear factor-?B essential modulator (NEMO) in a 9-month-old boy with ectodermal dysplasia with immunodeficiency who presented with methicillin-resistant Staphylococcus aureus subdural empyema. We performed in vitro experiments to determine if this novel mutation resulted in impaired NF-?B signaling.
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Endocytic pathway is required for Drosophila Toll innate immune signaling.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-19-2010
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The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.
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Anti-cancer effects of celecoxib on nasopharyngeal carcinoma HNE-1 cells expressing COX-2 oncoprotein.
Cytotechnology
PUBLISHED: 04-17-2010
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Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor with antitumor and antiangiogenic activities. To investigate the effects of celecoxib on nasopharyngeal carcinoma (NPC), HNE-1 cells were treated with celecoxib at various concentrations. MTT assay, migration assay and invasion assay were performed to observe the inhibitory activity of celecoxib on HNE-1 cells. Additionally, VEGF-A expression and radiation survival of NPC cell were also examined after treatment with celecoxib. Celecoxib treatment presented an anti-proliferation function in a time and dose-dependent manner on HNE-1 cells which highly express COX-2 protein. Celecoxib also displayed an obvious inhibitory activity on invasive capacity of NPC cells. Moreover, the celecoxibs effects to suppress VEGF-A expression and enhance radiosensitivity were detected in HNE-1 cells. These findings implicate that application of celecoxib may be an effective strategy for NPC therapy.
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SUMO, Ubiquitin, UBL Proteins: Implications For Human Diseases - Fifth International Conference.
IDrugs
PUBLISHED: 04-08-2010
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The fifth international conference on SUMO, Ubiquitin, UBL Proteins: Implications for Human Diseases, held in Houston, included topics covering the latest advances and new targets in the field of protein modification. This conference report highlights selected presentations on the structural characterization of ubiquitination and SUMOylation machinery; the regulation of ubiquitination enzymes, including E3 ligases; the functions and mechanism of action of SUMO-targeted ubiquitin ligases (STUbLs); the regulation of gene expression by SUMO; non-degradative functions of ubiquitin and SUMO in signal transduction; mechanisms and functions of ISG15 conjugation; the interaction of pathogens with host cell SUMOylation machinery; and stabilization of the Axin protein. Investigational drugs discussed include MLN-4924 (Millennium Pharmaceuticals Inc).
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NLRC5 negatively regulates the NF-kappaB and type I interferon signaling pathways.
Cell
PUBLISHED: 02-22-2010
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Stringent control of the NF-kappaB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-kappaB-dependent responses by interacting with IKKalpha and IKKbeta and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-kappaB and its responsive genes, TNF-alpha and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-kappaB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity.
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Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.
Cell
PUBLISHED: 02-22-2010
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RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.
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Peroxisomes are signaling platforms for antiviral innate immunity.
Cell
PUBLISHED: 02-16-2010
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Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.
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Comparison between two kinds of cigarette smoke condensates (CSCs) of the cytogenotoxicity and protein expression in a human B-cell lymphoblastoid cell line using CCK-8 assay, comet assay and protein microarray.
Mutat. Res.
PUBLISHED: 01-31-2010
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The differences of the cytogenotoxicity and proteins expression of human B-cell lymphoblastoid cells exposed to cigarette smoke condensates (CSCs) from two kinds of cigarettes were detected with CCK-8 assay, comet assay, protein microarray and western blot assay in vitro. Human B-cell lymphoblastoid cell line was exposed to CSCs from two cigarettes (which delivers approximately 3mg tar, 0.3mg nicotine, 3mg CO per cigarette for cigarette 1 and 15mg tar, 1.3mg nicotine, 15mg CO per cigarette for cigarette 2), and the exposure doses were 2.5, 5.0, 7.5, 10.0 and 12.5x10(-3)cigarettes/ml of CSCs for 24h in CCK-8 assay, 6.0, 8.0, 10.0, 12.0 and 14.0x10(-3)cigarettes/ml of CSCs for 4h in comet assay, and 10.0x10(-3)cigarettes/ml of CSCs for 4h in protein levels analysis. The results of CCK-8 assay and comet assay in the present study suggested that the cytogenotoxicity in cigarette 2 group was significantly higher than that in cigarette 1 group. The results of protein microarray and western blot assay showed that there were the differences of the expression levels of four proteins (i.e., RAR-beta, 14-3-3 sigma, XPF, and p57(Kip2) Ab-7) between cigarette 1 group and cigarette 2 group. Hence, it is possible that the RAR-beta, 14-3-3 sigma, XPF, and p57(Kip2) Ab-7 proteins serve as the molecular biomarkers in studying the cytogenotoxicity induced by CSCs.
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ATM- and NEMO-dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in response to genotoxic stress.
Mol. Cell
PUBLISHED: 01-12-2010
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Activation of the transcription factor NF-?B by multiple genotoxic stimuli modulates cancer cell survival. This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic I?B kinase (IKK); however, the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKK?/NEMO and ELKS (a protein rich in glutamate, leucine, lysine, and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM- and NEMO-dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-?B activation following genotoxic stimuli.
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Act1, a U-box E3 ubiquitin ligase for IL-17 signaling.
Sci Signal
PUBLISHED: 10-15-2009
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Interleukin-17 (IL-17), a proinflammatory cytokine mainly produced by cells of the T helper 17 (T(H)17) lineage, is required for host defense against bacterial and fungal infections and plays a critical role in the pathogenesis of inflammatory and autoimmune diseases. Act1 is an essential adaptor molecule in IL-17-mediated signaling and is recruited to the IL-17 receptor (IL-17R) upon IL-17 stimulation through an interaction between its SEFIR domain and that of the IL-17R. Here, we report that Act1 is a U-box E3 ubiquitin ligase and that its activity is essential for IL-17-mediated signaling pathways. Through the use of the Ubc13-Uev1A E2 complex, Act1 mediated the lysine-63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6), a component of IL-17-mediated signaling. Deletion and point mutations of the Act1 U-box abolished Act1-mediated ubiquitination of TRAF6 and impaired the ability of Act1 to restore IL-17-dependent signaling and expression of target genes in Act1(-/-) mouse embryonic fibroblasts. We also showed that the lysine-124 residue of TRAF6 was critical for efficient Act1-mediated ubiquitination of TRAF6 and for the ability of TRAF6 to mediate IL-17-induced activation of nuclear factor kappaB. Thus, we propose that Act1 mediates IL-17-induced signaling pathways through its E3 ubiquitin ligase activity and that TRAF6 is a critical substrate of Act1, which indicates the importance of protein ubiquitination in the IL-17-dependent inflammatory response.
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Core verbal working-memory capacity: the limit in words retained without covert articulation.
Q J Exp Psychol (Hove)
PUBLISHED: 09-03-2009
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Verbal working memory may combine phonological and conceptual units. We disentangle their contributions by extending a prior procedure (Chen & Cowan, 2005) in which items recalled from lists of previously seen word singletons and of previously learned word pairs depended on the list length in chunks. Here we show that a constant capacity of about 3 chunks holds across list lengths and list types, provided that covert phonological rehearsal is prevented. What remains is a core verbal working-memory capacity.
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How verbal memory loads consume attention.
Mem Cognit
PUBLISHED: 08-15-2009
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According to a traditional assumption about working memory, participants retain a series of verbal items for immediate recall using covert verbal rehearsal, without much need for attention. We reassessed this assumption by imposing a speeded, nonverbal choice reaction time (CRT) task following the presentation of each digit in a list to be recalled. When the memory load surpassed a few items, performance on the speeded CRT task became increasingly impaired. This CRT task impairment depended only on attention-related components of working memory; it was not alleviated by the presence of an auditory memory trace that automatically helped the recall of items at the ends of spoken lists. We suggest that attention-demanding refreshing of verbal stimuli occurs along with any covert rehearsal.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.