Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.

Relationships of Scopulariopsis based on LSU rDNA sequences.

Relationships between Scopulariopsis species and allied fungi were studied by sequencing a 350 bp gene region of the large subunit ribosomal RNA gene (LSU rDNA). In addition, a limited morphological dataset of nine characters was included in the cladistic analysis. Nineteen mitosporic strains (nine Scopulariopsis, five Wardomyces, three Doratomyces, one Trichurus and one Scedosporium species) and 21 meiosporic strains (14 Microascus, 4 Kernia and 3 Pithoascus species) were studied. The data were analysed using parsimony methods. Based on the analyses, the studied microascaceous fungi are divided to 12 molecular lineages. Most of the opportunistic human pathogenic Scopulariopsis species are placed in one clade ('Microascus manginii Clade'). Most synnematous anamorphs with Scopulariopsis-like conidia (Doratomyces and Trichurus) are placed in another clade ('Microascus albonigrescens Clade'), together with Wardomyces. Microascus sensu lato can be divided into seven clades which also incorporate all studied Pithoascus, Scopulariopsis, Wardomyces and Trichurus species and most of the Doratomyces species. Most of the Kernia teleomorphs and one Doratomyces species are placed in a different main clade, together with Pseudallescheria and Petriella. Future alternatives in the taxonomy of Microascus include splitting the genus or redefining it to include deviating taxa. More molecular data need to be obtained and considered in either case.

Striatal uptake of the dopamine reuptake ligand [11C]beta-CFT is reduced in Alzheimer's disease assessed by positron emission tomography.

Striatal dopamine reuptake sites were studied with PET in Alzheimer's disease (AD). A cocaine analogue, [11C]beta-CFT was used as a radioligand. In patients with AD, the reduction in [11C]beta-CFT uptake was about 20% from the age-adjusted mean value in control subjects, both in the putamen (p = 0.002) and in the caudate nucleus (p = 0.002). Thus, the putamen and the caudate nucleus were equally affected, in contrast to Parkinson's disease, which shows predominantly putaminal reduction. We found that the smaller the [11C]beta-CFT uptake in the putamen or in the caudate nucleus, the more severe the extrapyramidal symptoms. In healthy volunteers (nine women, six men; aged 23 to 70 years), [11C]beta-CFT uptake was reduced with age, both in the putamen (r = -0.70, p < 0.01) and in the caudate nucleus (r = -0.77, p < 0.001). The average decline per decade was 4.4% in the putamen and 4.7% in the caudate nucleus. We conclude that the brain dopaminergic system is affected in AD because the striatal uptake of the dopamine reuptake ligand [11C]beta-CFT is decreased. This reduction in [11C]beta-CFT uptake correlates with the severity of the extrapyramidal symptoms of the patients.