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    ABSTRACT: Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells-miR-142 and miR-223-and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth.
    The Journal of Immunology 11/2013;
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    ABSTRACT: Certain myrosinase-positive human gut bacteria can metabolize glucosinolates (GSLs) to produce isothiocyanates (ITC) as chemopreventive agents. We investigated glucoerucin, glucoiberin, and glucoraphanin (present in broccoli) metabolism by human gut strains. All tested bacteria metabolized glucoerucin to completion within 16 h to erucin and erucin nitrile (NIT). Lactobacillus agilis R16 metabolized only 10% of glucoiberin and glucoraphanin with no detectable products. Enterococcus casseliflavus CP1, however, metabolized 40-50% of glucoiberin and glucoraphanin producing relatively low concentrations of iberin and sulforaphane. Interestingly, Escherichia coli VL8 metabolized 80-90% of glucoiberin and glucoraphanin and also bioconverted glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively, producing erucin, erucin NIT, iberverin, and iberverin NIT from the two GSLs. The putative reductase enzyme in the cell-free extracts of this bacterium required both Mg(2+) and NAD(P)H as cofactors for bioconversion. The cell-free extract of E. coli VL8 containing the reductase enzyme was able to reduce both the GSL glucoraphanin and its hydrolysis product sulforaphane to glucoerucin and erucin/erucin NIT, respectively. The composition and metabolic activity of the human gut bacteria can indirectly impact on the potential chemopreventive effects of GSL-derived metabolites.
    Molecular Nutrition & Food Research 10/2013;
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    ABSTRACT: The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection LtpD localized to the cytoplasmic face of the membrane of the Legionella containing vacuole (LCV). In A549 lung epithelial cells, ectopically-expressed LtpD localized to large vesicular structures which contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17 kDa fragment, LtpD471-626, which was essential for targeting ectopically-expressed LtpD to vesicular structures and for association of translocated LtpD with the LCV. LtpD471-626 bound directly to phosphatidylinositol-3-phosphate (PtdIns (3)P) in vitro and co-localized with the PtdIns (3)P markers FYVE and SetA in co-transfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector, which has a role in intracellular bacterial replication.
    Infection and immunity 09/2013;
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    ABSTRACT: Ovarian cancer is associated with limited overall survival, due to problems in early detection and therapy. Membrane ion channels have been proposed to play a significant, concerted role in the cancer process, from initial proliferation to metastasis, and promise to be early, functional biomarkers. We review the evidence for ion channel and aquaporin expression and functioning in human ovarian cancer cells and tissues. In vitro, K(+) channels, mainly voltage-gated, including Ca(2+)-activated channels, have been found to control the cell cycle, as in other cancers. Voltage-gated, volume-regulated and intracellular Cl(-) channels have been detected in vitro and in vivo and shown to be involved in proliferation, adhesion and invasion. Evidence for 'transient receptor potential', voltage-gated sodium and calcium channels, which have been shown to contribute to pathogenesis of other carcinomas, is also emerging in ovarian cancer. Aquaporins may be involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. It is concluded that functional expression of ion channels and their regulation by steroid hormones and growth factors are an integral part of ovarian cancer development and progression. Furthermore, ion channels may be involved in multidrug resistance, commonly associated with treatment of ovarian cancer. We propose that ion channel studies can facilitate our understanding of the pathobiology of ovarian cancer and, ultimately, can serve as viable novel targets for its clinical management.
    European journal of cancer (Oxford, England: 1990) 05/2013;
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    ABSTRACT: Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacteria. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in haemocytes showed signs of instability and leakage. Furthermore, in contrast to wild type L. pneumophila, a ΔsdhA mutant caused a transient depletion of haemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.
    Infection and immunity 05/2013;
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    ABSTRACT: Rituximab, which binds CD20 on B cells, is one of the best characterised antibodies used in the treatment of B cell malignancies and autoimmune diseases. Rituximab triggers Natural Killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC - nor what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B cell surface, independent of antibody cross-linking or intercellular contact. Unexpectedly, other proteins, including ICAM-1 and moesin, were selectively recruited to the cap of CD20 and the MTOC became polarised towards the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarised compared to being un-polarised. Polarised B cells were lysed more frequently still, when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarisation of B cells and this augments its therapeutic function in triggering NK cell-mediated ADCC.
    Blood 04/2013;
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    ABSTRACT: We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases the core of the protein adopts a compact helical structure flanked by flexible N and C-termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases the VPg cores are stabilised by networks of hydrophobic and salt-bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilised the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined but knowledge of its structure will facilitate future investigations.
    Journal of Virology 03/2013;
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    ABSTRACT: Unusually for a eukaryote, Trypanosoma brucei transcribes its variant surface glycoprotein (VSG) gene expression sites (ESs) in a monoallelic fashion using RNA polymerase I (Pol I). It is still unclear how ES transcription is controlled in T. brucei. Here, we show that the TDP1 architectural chromatin protein is an essential high mobility group box (HMGB) protein facilitating Pol I transcription in T. brucei. TDP1 is specifically enriched at the active compared with silent VSG ES and immediately downstream of ribosomal DNA promoters and is abundant in the nucleolus and the expression site body subnuclear compartments. Distribution of TDP1 at Pol I-transcribed loci is inversely correlated with histones. Depletion of TDP1 results in up to 40-90% reduction in VSG and rRNA transcripts and a concomitant increase in histones H3, H2A and H1 at these Pol I transcription units. TDP1 shares features with the Saccharomyces cerevisiae HMGB protein Hmo1, but it is the first architectural chromatin protein facilitating Pol I-mediated transcription of both protein coding genes as well as rRNA. These results show that TDP1 has a mutually exclusive relationship with histones on actively transcribed Pol I transcription units, providing insight into how Pol I transcription is controlled.
    Nucleic Acids Research 01/2013;
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    ABSTRACT: Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca(2+) channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
    PLoS Pathogens 01/2013; 9(1):e1003145.
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    ABSTRACT: This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated μCT (DLIT-μCT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-μCT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-μCT imaging and bacterial enumeration from feces for 8 days. The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and μCT scans using a single imaging platform. The low dose μCT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.
    Journal of Visualized Experiments 01/2013;
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