Genetic variation and differentiation in captive and wild zebra finches (Taeniopygia guttata)

The zebra finch (Taeniopygia guttata) is a small Australian grassland songbird that has been domesticated over the past two centuries. Because it is easy to breed in captivity, it has become a widely used study organism, especially in behavioural research. Most work has been conducted on domesticated populations maintained at numerous laboratories in Europe and North America. However, little is known about the extent to which, during the process of domestication, captive populations have gone through bottlenecks in population size, leading to inbred and potentially genetically differentiated study populations. This is an important issue, because (i) behavioural studies on captive populations might suffer from artefacts arising from high levels of inbreeding or lack of genetic variation in such populations, and (ii) it may hamper the comparability of research findings. To address this issue, we genotyped 1000 zebra finches from 18 captive and two wild populations at 10 highly variable microsatellite loci. We found that all captive populations have lost some of the genetic variability present in the wild, but there is no evidence that they have gone through a severe bottleneck, as the average captive population still showed a mean of 11.7 alleles per locus, compared to a mean of 19.3 alleles/locus for wild zebra finches. We found significant differentiation between the captive populations (F(ST) = 0.062). Patterns of genetic similarity closely match geographical relationships, so the most pronounced differences occur between the three continents: Australia, North America, and Europe. By providing a tree of the genetic similarity of the different captive populations, we hope to contribute to a better understanding of variation in research findings obtained by different laboratories.     

Preparation of tetrahydroimidazo[2,1-a]isoquinolines and their use as inhibitors of gastric acid secretion

A series of novel tetrahydroimidazo[2,1-a]isoquinolines was prepared based on a hetero Diels–Alder reaction between an enamine and 1,2,4-triazine as key step. A structure–activity relationship was established focussing on the influence of the substitution pattern in position 3 and 6 of the heterocycle on antisecretory activity, lipophilicity, and pK_a value. Potent inhibitors of the gastric acid pump were identified.     

Combining immunological and molecular data to assess phylogenetic relations of some Greek Podarcis species

Most recent molecular studies revealed the phylogeny of Greek Podarcis species, which for years remained elusive, due to discordant data produced from various chromosomal, complement fixation and protein studies. In this report, we analyzed cellular immune responses of spleen-derived lymphocytes from six allopatric Podarcis species encountered in Greece, by assessing two-way mixed lymphocyte reaction (MLR)-induced proliferation. On the basis of stimulation indices (S.I.) as determined from cultures set up from xenogeneic splenocytes coincubated in pairs, we generated a phylogenetic tree, fully consistent with the phylogenetic relationships of Podarcis as determined by parallel analyses based on partial mitochondrial (mt) DNA sequences. Although the exact mechanisms triggering lymphocyte responses in lizard two-way xenogeneic MLR are not fully understood, our results show the potential use of cell-mediated immune responses as an additional approach to mtDNA analysis, for species delimitation within specific lizard taxa.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Semi‐dry grasslands along a climatic gradient across Central Europe: Vegetation classification with validation

Question: What is the variation in species composition of Central European semi‐dry grasslands? Can we apply a training‐and‐test validation approach for identifying phytosociological associations which are floristically well defined in a broad geographic comparison; can we separate them from earlier described associations with only a local validity? Location: A 1200 km long transect running along a gradient of increasing continentality from central Germany via Czech Republic, Slovakia, NE Austria, Hungary to NW Romania. Methods: Relevés with > 25% cover of Brachypodium pin‐natum and/or Bromus erectus were geographically selected from a larger database. They were randomly split into two data sets, TRAINING and TEST, each with 422 relevés. Cluster analysis was performed for each data set on scores from significant principal coordinates. Different partitions of the TRAINING data set were validated on the TEST data set, using a new method based on the comparison of % frequencies of species occurrence in clusters. Clusters were characterized by statistically defined groups of diagnostic species and values of climatic variables. Results: Species composition changed along the NW‐SE gradient and valid clusters were geographically well separated. Optimal partition level was at 11 clusters, six being valid: two clusters Germany and the Czech Republic corresponded to the Bromion erecti; two clusters from the Czech Republic and Hungary to the Cirsio‐Brachypodion, and two clusters were transitional between these two alliances. Conclusion: The training‐and‐test validation method used in this paper proved to be efficient for discriminating between robust clusters, which are appropriate candidates for inclusion in the national or regional syntaxonomic overviews, and weak clusters, which are specific to the particular classification of the given data set.     

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.     

High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

A novel cold-inducible expression system for Bacillus subtilis

Production of recombinant proteins at low temperatures is one strategy to prevent formation of protein aggregates and the use of an expensive inducer such as IPTG. We report on the construction of two expression vectors both containing the cold-inducible des promoter of Bacillus subtilis, where one allows intra- and the other extracellular synthesis of recombinant proteins. Production of recombinant proteins started within the first 30min after temperature downshock to 25 degrees C and continued for about 5h.     

Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.