Genomic heterogeneity of small ruminant lentiviruses detected by PCR

In order to detect a large spectrum of small ruminant lentiviruses, primers for PCR were chosen in conserved parts of the LTR and GAG genes of Icelandic Visna virus 1514 and of the POL gene of caprine arthritis-encephalitis virus. This set of primers was tested in six different caprine arthritis-encephalitis virus (CAEV)- and Maedi-Visna virus isolates of Dutch, American and Swiss origin. The LTR primers allowed the detection of the corresponding fragments of all isolates. The GAG primers allowed amplification of the corresponding fragments of all but the Swiss Maedi-Visna virus strain OLV. Using the POL primers, one Maedi-Visna- and two caprine arthritis-encephalitis virus strains were detected after one round of amplification. Sequencing of the GAG and POL amplification products and comparison to Icelandic Visna virus and CAEV strain CO revealed total heterogeneity of 38% for the GAG- and 28% for the POL fragment. The virus strains studied fall into two groups which are more closely related to one another than to Icelandic Visna virus.

Interaction of small molecules with specific immune receptors: the p-i concept and its consequences

Drugs may stimulate the immune system by forming stable new antigenic complexes consisting of the drug or drug metabolite which is covalently bound to a protein or peptide (hapten-carrier complex). Both, B- and T-cell immunity may arise, the latter directed to hapten modified peptides presented by HLA molecules. Beside this immunological stimulation, drugs can also stimulate the immune system through binding by non-covalent bonds to proteins like immune receptors. This so-called “pharmacological interaction with immune receptors” concept (“p-i concept”) may occur with HLA or TCR molecules themselves (p-i HLA or p-i TCR), and not the immunogenic peptide. It is a type of “off-target” activity of the drug on immune receptors, but more complex as various cell types, cell interactions and functionally different T cells are involved. In this review the conditions which lead to activation of T cells by p-i are discussed: important factors for a functional consequence of drug binding is the location of binding (p-i HLA or p-i TCR); the exact site within these immune receptors; the affinity of binding and the finding that p-i HLA can stimulate the immune system like an allo-allele. The p-i concept is able to solve some puzzles of drug hypersensitivity reactions and are a basis to better treat and potentially avoid drug hypersensitivity reactions. Moreover, the p-i concept shows that in contrast to previous beliefs small molecules do interact with immune receptors with functional consequence. But these interactions are not based on “immune recognition”, are at odds with some immunological concepts, but may nevertheless open new possibilities to understand and even treat immune reactions

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

The RNome of a cell is highly diverse and consists besides messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) also of other small and long transcript entities without apparent coding potential. This class of molecules, commonly referred to as non-protein-coding RNAs (ncRNAs), is involved in regulating numerous biological processes and thought to contribute to cellular complexity. Therefore, much effort is put into their identification and further functional characterization. Here we provide a cost-effective and reliable method for cDNA library construction of small RNAs in the size range of 20-500 residues. The effectiveness of the described method is demonstrated by the analysis of ribosome-associated small RNAs in the eukaryotic model organism Trypanosoma brucei.

Ready for a crisis? How supervisory boards affect the formalized crisis procedures of small and medium-sized family firms in Germany

Overcoming a crisis situation in which the socioemotional wealth (SEW) of a family is at risk can be threatened by a lack of formal crisis procedures, which can increase the probability of organizational decline. Thus, not being prepared for a crisis situation may be a critical factor in the long-term survival of family firms. From a corporate governance perspective, supervisory boards may achieve higher levels of crisis readiness. Applying the resourced-based view and SEW theory, we analyze the relationship between family ownership and formalized crisis procedures in 150 small and medium-sized German firms. Our results show that formalized crisis procedures decrease as family ownership increases. Including supervisory boards in our analysis, we find a significant moderating effect of supervisory boards on the relationship between family ownership and formalized crisis procedures. Specifically, our results suggest that family firms with supervisory boards show similar levels of formalized crisis procedures as non-family firms with supervisory boards. In contrast, family firms without supervisory boards exhibit lower levels of formalized crisis procedures compared with non-family firms without supervisory boards. We also discuss managerial implications, limitations, and future research.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25–55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38–75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.

Narrow dust jets in a diffuse gas coma: a natural product of small active regions on comets

Comets often display narrow dust jets but more diffuse gas comae when their eccentric orbits bring them into the inner solar system and sunlight sublimates the ice on the nucleus. Comets are also understood to have one or more active areas covering only a fraction of the total surface active with sublimating volatile ices. Calculations of the gas and dust distribution from a small active area on a comet’s nucleus show that as the gas moves out radially into the vacuum of space it expands tangentially, filling much of the hemisphere centered on the active region. The dust dragged by the gas remains more concentrated over the active area. This explains some puzzling appearances of comets having collimated dust jets but more diffuse gaseous atmospheres. Our test case is 67P/Churyumov–Gerasimenko, the Rosetta mission target comet, whose activity is dominated by a single area covering only 4% of its surface.