The prion organotypic slice culture assay--POSCA

Methods enabling prion replication ex vivo are important for advancing prion science. However, few such technologies exist and many prion strains are intractable with them. Here, we describe a prion organotypic slice culture assay (POSCA), which allows for prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Organotypic slices are incubated with infectious inoculum as free-floating sections, washed and cultured for up to 8 weeks. Slice cultures are a rich source of protein or RNA and allow for stringent comparisons between uninfected and prion-infected samples generated from the same mouse. Thirty-five days after contact with prions, cerebellar slices have amplified PrP(Sc) quantitatively similar to that seen in vivo, but accelerated fivefold. The POSCA detects replication of specific prion strains from disparate sources, including bovines and ovines, with variable efficiency. The culture procedure and prion infection can be performed in 8 h.     

Mutation of miRNA target sequences during human evolution

It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.     

Evaluation of the redox dye 5-cyano-2,3-tolyl-tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries

A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries.     

Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.     

Balanced harvest: concept,policies, evidence,and management implications

Reviews in Fish Biology and Fisheries - Balanced harvest has been proposed to reduce fishing impact on ecosystems while simultaneously maintaining or even increasing fishery yield. The concept has...     

Endogenous Interferon-β-Inducible Gene Expression and Interferon-β-Treatment Are Associated with Reduced T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive CD4⁺ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4⁺ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4⁺ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4⁺ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4⁺ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.