miRNA-mRNA integrated analysis reveals roles for miRNAs in primary breast tumors

Introduction

Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.

Methods

We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods.

Results

We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process.

Conclusion

This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana.     

Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism

Although it is known that septic shock induces immunosuppression, the mechanism for this phenomenon is not well understood. Monocytes play a central role in septic shock pathophysiology, which is also characterized by an increased proportion of natural regulatory T (Treg) cells. We therefore investigated whether Treg could be involved in the decreased monocyte expression of CD14 and HLA-DR observed during septic shock. We demonstrated that human Treg inhibit LPS-induced retention of monocyte CD14. Because loss of CD14 is a hallmark of monocyte apoptosis, this suggests that Treg inhibit monocyte survival. This effect was largely mediated through the release of a soluble mediator that was not identical with either IL-10 or IL-4. The Fas/FasL pathway participated in the effect as it was blocked by anti-FasL Abs and reproduced by Fas agonist and recombinant soluble FasL. Furthermore, expression of FasL was much higher on Treg than on their CD25(-) counterparts. Collectively, these results indicate that Treg act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism involving the Fas/FasL pathway. This may be an important mechanism for septic shock-induced immunosuppression and may offer new perspectives for the treatment of this deadly disease.     

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

Gene network reconstruction identifies the authentic trans-prenyl diphosphate synthase that makes the solanesyl moiety of ubiquinone-9 in Arabidopsis

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate--product of gene At2g34630--that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main--if not sole--contributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.     

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA)

The chlorophyll-protein CP43′ (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the ‘energy-dependent non-photochemical quenching’ (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.     

Enzyme phylogenies as markers for the oxidation state of the environment: The case of respiratory arsenate reductase and related enzymes

Background  

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far.