An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Taxonomic distribution and origins of the extended LHC (light-harvesting complex) antenna protein superfamily

Background  

The extended light-harvesting complex (LHC) protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS). The evolution of this complex superfamily has long remained elusive, partially due to previously missing families.     

Optimum allocation of conservation funds and choice of conservation programs for a set of African cattle breeds

Although funds for livestock conservation are limited there is little known about the optimal allocation of conservation funds. A new algorithm was used to allocate Mio US$ 1, 2, 3, 5 or unlimited funds, discounted over 50 years, on 23 African cattle breeds conserved with four different possible conservation programs. Additionally, Mio US$ 1 was preferably allocated to breeds with special traits. The conceptional in situ conservation programs strongly involve breeders and give them part of the responsibility for the conservation of the breed. Therefore, the pure in situ conservation was more efficient than cryoconservation or combined in situ and cryoconservation. The average annual discounted conservation cost for a breed can be as low as US$ 1000 to US$ 4400 depending on the design of the conservation program and the economic situation of the country of conservation. The choice of the breeds and the optimal conservation program and the amount of money allocated to each breed depend on many factors such as the amount of funds available, the conservation potential of each breed, the effects of the conservation program as well as its cost. With Mio US$ 1, 64% of the present diversity could be maintained over 50 years, which is 13% more than would be maintained if no conservation measures were implemented. Special traits could be conserved with a rather small amount of the total funds. Diversity can not be conserved completely, not even with unlimited funds. A maximum of 92% of the present diversity could be conserved with Mio US$ 10, leaving 8% of the diversity to unpredictable happenings. The suggested algorithm proved to be useful for optimal allocation of conservation funds. It allocated the funds optimally among breeds by identifying the most suited conservation program for each breed, also accounting for differences in currency exchange rates between the different countries.     

A Scale-Corrected Comparison of Linkage Disequilibrium Levels between Genic and Non-Genic Regions

The understanding of non-random association between loci, termed linkage disequilibrium (LD), plays a central role in genomic research. Since causal mutations are generally not included in genomic marker data, LD between those and available markers is essential for capturing the effects of causal loci on localizing genes responsible for traits. Thus, the interpretation of association studies requires a detailed knowledge of LD patterns. It is well known that most LD measures depend on minor allele frequencies (MAF) of the considered loci and the magnitude of LD is influenced by the physical distances between loci. In the present study, a procedure to compare the LD structure between genomic regions comprising several markers each is suggested. The approach accounts for different scaling factors, namely the distribution of MAF, the distribution of pair-wise differences in MAF, and the physical extent of compared regions, reflected by the distribution of pair-wise physical distances. In the first step, genomic regions are matched based on similarity in these scaling factors. In the second step, chromosome- and genome-wide significance tests for differences in medians of LD measures in each pair are performed. The proposed framework was applied to test the hypothesis that the average LD is different in genic and non-genic regions. This was tested with a genome-wide approach with data sets for humans (Homo sapiens), a highly selected chicken line (Gallus gallus domesticus) and the model plant Arabidopsis thaliana. In all three data sets we found a significantly higher level of LD in genic regions compared to non-genic regions. About 31% more LD was detected genome-wide in genic compared to non-genic regions in Arabidopsis thaliana, followed by 13.6% in human and 6% chicken. Chromosome-wide comparison discovered significant differences on all 5 chromosomes in Arabidopsis thaliana and on one third of the human and of the chicken chromosomes.     

Assessing the degree of stratification between closely related Holstein-Friesian populations

Genomic information is an important part of the routine evaluation of dairy cattle and provides the wide availability of animals genotyped using single nucleotide polymorphism (SNP) microarrays. We analyzed 2243 Polish and 2294 German Holstein-Friesian bulls genotyped using the Illumina BovineSNP50 BeadChip. For each bull, estimated breeding values (EBVs) calculated from national routine genetic evaluation were available for production traits and for somatic cell score (SCS). Separately for each population, we estimated SNP haplotypes, pairwise linkage disequilibrium (LD), and SNP effects. The SNP genetic covariance between both populations was estimated using a bivariate mixed model. The average LD was lower in the Polish than in the German population and, with increasing genomic distance, LD decays 1.7 times more rapidly in German than in Polish cattle. The comparison of SNP allele frequencies for base populations estimated separately using Polish and German data revealed a very good agreement. The comparison of genetic effects corresponding to various window lengths defined in bp emerged a systematic pattern: regardless of the length of the compared region, few significant differences were found for production traits, while many were observed for SCS. For each trait, the German population had much higher SNP variances than the Polish population and the genetic covariance estimates were all positive. Depending on traits’ inheritance mode, the additive genetic variation can be stored in many genes following the infinitesimal model (like for SCS) or distributed between genes with high effects and the polygenic “background” (like for production traits). Accounting for those differences has implications on the prospective international genomic evaluation.     

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel,potent and selective GABAA α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA_A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.     

Diversity and evolution of repABC type plasmids in Rhodobacterales

The repABC operon is the prevalent replication unit of alphaproteobacterial plasmids. Their semi-autonomy is ensured by the essential replicase gene repC as well as the repAB partitioning cassette. While conserved repAB modules are widespread among bacterial plasmids and homologues are even responsible for chromosome partitioning, repC genes are exclusively present in Alphaproteobacteria . RepABC operons contain two strong incompatibility regions, namely a small regulative antisense RNA gene ( incα ) and a palindromic centromere region ( incβ ), which were previously used to classify these replicons. The present survey pursued a complementary strategy essentially following the rationale that all plasmids identified from a single bacterium are per se compatible. We established a novel classification scheme for plasmids based on comprehensive phylogenetic analyses of repC , repA and repB genes. Our case study is focused on the Roseobacter clade ( Rhodobacterales ), one of the most successful lineages of the marine bacterioplankton. Its global significance was shown in several studies and the interest in these organisms is reflected by more than 40 upcoming genome projects. Based on phylogenetic RepC analyses we identified nine compatibility groups that are expected to stably coexist within the same cell. This prediction is supported by RepA and RepB phylogenies, moreover independent evidence is delivered by the group specificity of highly conserved palindromes ( incβ ).     

Genetic analysis of reactivity to humans in Goettingen minipigs

Goettingen minipigs are laboratory animals with an increasing demand over the last few years. At the moment, Goettingen minipigs are not selected for a low reactivity to humans and this trait is not included in the breeding programme. However, it is obvious that there is a need for genetically non-responding minipigs during handling to facilitate the treatment and restraint of the animals which is often needed in biomedical experiments. A first testing scheme was developed to evaluate the reactivity of Goettingen minipigs to humans and to analyse whether the trait reactivity to humans can be considered in the breeding programme. In this study temperament scores of this testing scheme for nine different traits from 10,033 animals collected from 2005 to 2008 were analysed. Temperament was subjectively scored on a scale from 1 to 5 while the pig is caught (C), held on the arms (A), standing in a box for weighing (W), standing on a table (T) and walking on the ground (G). The traits were a combination of these situations evaluated at three different ages (2, 4 and 6 months). Genetic parameters were estimated using bivariate models and different possible selection strategies were examined. Heritabilities were low to moderate with a range from 0.09 to 0.22 and phenotypic and genetic correlations between the nine traits were moderate to high with phenotypic correlations between 0.12 (W2 and G4) and 0.64 (W2 and A2) and genetic correlations between 0.44 (A4 and C6) and 1.00 (e.g. W2 and A4). It was shown that the highest genetic progress per year can be obtained when all nine traits are considered in the selection index. Under an economical point of view the selection on the basis of the two arm traits plus the trait W2 should be preferred.Based on a critical discussion of the explanatory power of the used scoring system a new evaluation scheme was developed. In this scheme the minipigs can be divided into responding and non-responding animals whereas the latter are desired for selection. The suggested scoring system offers better possibilities for statistical analyses. It is planned to include the selection for non-responding Goettingen minipigs in the routine breeding programme.