Circulating miRNA‐375 as a potential novel biomarker for active Kaposi’s sarcoma in AIDS patients

The aim of this study was to identify circulating microRNAs (miRNAs) that could be used as biomarkers in patients at risk for or affected by AIDS‐Kaposi's sarcoma (KS). Screening of 377 miRNAs was performed using low‐density arrays in pooled plasma samples of 10 HIV/human herpesvirus 8 (HHV8)‐infected asymptomatic and 10 AIDS‐KS patients before and after successful combined antiretroviral therapy (cART). MiR‐375 was identified as a potential marker of active KS, being the most down‐regulated in AIDS‐KS patients after cART and the most up‐regulated in naïve AIDS‐KS patients compared to naïve asymptomatic subjects. Validation on individual plasma samples confirmed that miR‐375 levels were higher in AIDS‐KS compared to asymptomatic patients, decreased after cART‐induced remission in most AIDS‐KS patients and increased in patients with active KS. In asymptomatic patients miR‐375 was up‐regulated after cART in both screening and validation. Statistical analyses revealed an association between miR‐375 changes and CD4 cell counts, which could explain the discordant cases and the opposite trend between asymptomatic and AIDS‐KS patients. These data suggest that circulating miR‐375 might be a good indicator of active AIDS‐KS. Moreover, changes in miR‐375 levels may have a prognostic value in HIV/HHV8‐infected patients undergoing treatment. Further large‐scale validation is needed.     

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.     

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.     

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.     

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.     

Developing Native Multispecies Sod: An Alternative Rehabilitation Method for Disturbed Lands

Natural and anthropogenic disturbance provides a mechanism for resetting the species assemblage within natural communities. Following anthropogenic disturbance particularly, there are generally more weedy species. Restoration of ecosystem structure and function in disturbed areas could aid in the control of these undesirable species. Attempts to re‐establish vegetation via direct sowing, hydroseeding, drill seeding, imprinting or plugging of either native or non‐native species can be effective, but can result in increased erosion and weed proliferation prior to desirable cover of the intended species. The use of native species for rehabilitation can be preferable because non‐native species are often less suited to the local environment and can pose a threat to the integrity of adjacent ecosystems. Native multispecies sod could become a useful rehabilitation tool, but research is needed to determine which species to use and the appropriate number of species to include. Mixtures of 4–7 native species were grown for 7 months, and clipped dry biomass, species percent abundance, and total ground cover were assessed over time in order to determine the suitability of 20 native species for sod production. Despite differences between mixtures at some harvests, all mixtures established and produced similar biomass by the end of the study. Final percent cover varied widely among species; 16 of the 20 species increased significantly over the course of production. Differences in total ground cover between mixtures were explained by the growth of the particular species rather than any synergistic effect of species diversity.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.