The identification of neurotensin NTS1 receptor partial agonists through a ligand-based virtual screening approach

We identified small molecule NTS1R agonist compounds through virtual screening of the corporate database using a ROCS approach that searches multi-conformer representations efficiently. As a starting point for the ROCS search, we used the known NTS1R selective antagonist, SR-48527, based on the hypothesis that NT agonists and antagonists might share similar binding regions. Conformations were expanded and selected as database search queries based on a cluster analysis. The search provided us with virtual hits that were tested in intracellular calcium mobilization assays of NTS1R agonist and antagonist activities measured in FLIPR format as well as in [(3)H]NT competition binding studies. The results indicated that two initial hits produced partial agonist activity with potency in the moderate micromolar range.     

Imputation of high-density genotypes in the Fleckvieh cattle population

Background

Currently, genome-wide evaluation of cattle populations is based on SNP-genotyping using ~ 54 000 SNP. Increasing the number of markers might improve genomic predictions and power of genome-wide association studies. Imputation of genotypes makes it possible to extrapolate genotypes from lower to higher density arrays based on a representative reference sample for which genotypes are obtained at higher density.

Methods

Genotypes using 639 214 SNP were available for 797 bulls of the Fleckvieh cattle breed. The data set was divided into a reference and a validation population. Genotypes for all SNP except those included in the BovineSNP50 Bead chip were masked and subsequently imputed for animals of the validation population. Imputation of genotypes was performed with Beagle, findhap.f90, MaCH and Minimac. The accuracy of the imputed genotypes was assessed for four different scenarios including 50, 100, 200 and 400 animals as reference population. The reference animals were selected to account for 78.03%, 89.21%, 97.47% and > 99% of the gene pool of the genotyped population, respectively.

Results

Imputation accuracy increased as the number of animals and relatives in the reference population increased. Population-based algorithms provided highly reliable imputation of genotypes, even for scenarios with 50 and 100 reference animals only. Using MaCH and Minimac, the correlation between true and imputed genotypes was > 0.975 with 100 reference animals only. Pre-phasing the genotypes of both the reference and validation populations not only provided highly accurate imputed genotypes but was also computationally efficient. Genome-wide analysis of imputation accuracy led to the identification of many misplaced SNP.

Conclusions

Genotyping key animals at high density and subsequent population-based genotype imputation yield high imputation accuracy. Pre-phasing the genotypes of the reference and validation populations is computationally efficient and results in high imputation accuracy, even when the reference population is small.     

Limited carbon and mineral nutrient gain from mycorrhizal fungi by adult Australian orchids

? Premise of the study: In addition to autotrophic and fully mycoheterotrophic representatives, the orchid family comprises species that at maturity obtain C and N partially from fungal sources. These partial mycoheterotrophs are often associated with fungi that simultaneously form ectomycorrhizas with trees. This study investigates mycorrhizal nutrition for orchids from the southwestern Australian biodiversity hotspot. ? Methods: The mycorrhizal fungi of 35 green and one achlorophyllous orchid species were analyzed using molecular methods. Nutritional mode was identified for 27 species by C and N isotope abundance analysis in comparison to non-orchids from the same habitat. As a complementary approach, (13)CO(2) pulse labeling was applied to a subset of six orchid species to measure photosynthetic capacity. ? Key results: Almost all orchids associated with rhizoctonia-forming fungi. Due to much higher than expected variation within the co-occurring nonorchid reference plants, the stable isotope approach proved challenging for assigning most orchids to a specialized nutritional mode; therefore, these orchids were classified as autotrophic at maturity. The (13)CO(2) pulse labeling confirmed full autotrophy for six selected species. Nonetheless, at least three orchid species (Gastrodia lacista, Prasophyllum elatum, Corybas recurvus) were identified as nutritionally distinctive from autotrophic orchids and reference plants. ? Conclusions: Despite the orchid-rich flora in southwestern Australia, partial mycoheterotrophy among these orchids is less common than in other parts of the world, most likely because most associate with saprotrophic fungi rather than ectomycorrhizal fungi.     

Genome-wide association study identifies two major loci affecting calving ease and growth-related traits in cattle

Identifying quantitative trait loci (QTL) underlying complex, low-heritability traits is notoriously difficult. Prototypical for such traits, calving ease is an important breeding objective of cattle (Bos taurus)-improving programs. To identify QTL underlying calving ease, we performed a genome-wide association study using estimated breeding values (EBVs) as highly heritable phenotypes for paternal calving ease (pCE) and related traits. The massively structured study population consisted of 1800 bulls of the German Fleckvieh (FV) breed. Two pCE-associated regions on bovine chromosomes (BTA) 14 and 21 (P = 5.72 × 10(-15) and P = 2.27 × 10(-8), respectively) were identified using principal components analysis to correct for population stratification. The two most significantly associated SNPs explain 10% of the EBV variation. Since marker alleles with negative effect on pCE have positive effects on growth-related traits, the QTL may exert their effects on the birthing process through fetal growth traits. The QTL region on BTA14 corresponds to a human chromosome (HSA) region that is associated with growth characteristics. The HSA region corresponding to the BTA21 pCE QTL is maternally imprinted and involved in the Prader-Willi and Angelman syndromes. Resequencing of positional candidate genes on BTA14 revealed a highly significantly (P = 1.96 × 10(-14)) associated polymorphism ablating a polyadenylation signal of the gene encoding ribosomal protein S20 (RPS20). Our study demonstrates the leverage potential of EBVs in unraveling the genetic architecture of lowly heritable traits.     

Usefulness of Multiparental Populations of Maize (Zea mays L.) for Genome-Based Prediction

The efficiency of marker-assisted prediction of phenotypes has been studied intensively for different types of plant breeding populations. However, one remaining question is how to incorporate and counterbalance information from biparental and multiparental populations into model training for genome-wide prediction. To address this question, we evaluated testcross performance of 1652 doubled-haploid maize (Zea mays L.) lines that were genotyped with 56,110 single nucleotide polymorphism markers and phenotyped for five agronomic traits in four to six European environments. The lines are arranged in two diverse half-sib panels representing two major European heterotic germplasm pools. The data set contains 10 related biparental dent families and 11 related biparental flint families generated from crosses of maize lines important for European maize breeding. With this new data set we analyzed genome-based best linear unbiased prediction in different validation schemes and compositions of estimation and test sets. Further, we theoretically and empirically investigated marker linkage phases across multiparental populations. In general, predictive abilities similar to or higher than those within biparental families could be achieved by combining several half-sib families in the estimation set. For the majority of families, 375 half-sib lines in the estimation set were sufficient to reach the same predictive performance of biomass yield as an estimation set of 50 full-sib lines. In contrast, prediction across heterotic pools was not possible for most cases. Our findings are important for experimental design in genome-based prediction as they provide guidelines for the genetic structure and required sample size of data sets used for model training.     

Classic Selective Sweeps Revealed by Massive Sequencing in Cattle

Human driven selection during domestication and subsequent breed formation has likely left detectable signatures within the genome of modern cattle. The elucidation of these signatures of selection is of interest from the perspective of evolutionary biology, and for identifying domestication-related genes that ultimately may help to further genetically improve this economically important animal. To this end, we employed a panel of more than 15 million autosomal SNPs identified from re-sequencing of 43 Fleckvieh animals. We mainly applied two somewhat complementary statistics, the integrated Haplotype Homozygosity Score (iHS) reflecting primarily ongoing selection, and the Composite of Likelihood Ratio (CLR) having the most power to detect completed selection after fixation of the advantageous allele. We find 106 candidate selection regions, many of which are harboring genes related to phenotypes relevant in domestication, such as coat coloring pattern, neurobehavioral functioning and sensory perception including KIT, MITF, MC1R, NRG4, Erbb4, TMEM132D and TAS2R16, among others. To further investigate the relationship between genes with signatures of selection and genes identified in QTL mapping studies, we use a sample of 3062 animals to perform four genome-wide association analyses using appearance traits, body size and somatic cell count. We show that regions associated with coat coloring significantly (P<0.0001) overlap with the candidate selection regions, suggesting that the selection signals we identify are associated with traits known to be affected by selection during domestication. Results also provide further evidence regarding the complexity of the genetics underlying coat coloring in cattle. This study illustrates the potential of population genetic approaches for identifying genomic regions affecting domestication-related phenotypes and further helps to identify specific regions targeted by selection during speciation, domestication and breed formation of cattle. We also show that Linkage Disequilibrium (LD) decays in cattle at a much faster rate than previously thought.     

D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone

Schizophrenia is a highly heritable neuropsychiatric disorder affecting ～1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.