The alpha7 nicotinic acetylcholine receptor (nAChR) is a potential target in neuroinflammation. Screening a plant extract library identified Solidago nemoralis as containing methyl-quercetin derivatives that are relatively selective ligands for the alpha7 nAChR. Flavonoids are not known for this activity, so we screened a small library of pure flavonoids to confirm our findings. Some flavonoids, e.g. rhamnetin, displaced a selective alpha7 nAChR radioligand from rat brain membranes whereas similar structures e.g. sakuranetin, did not. To evaluate the contribution of this putative nAChR activity to the known anti-inflammatory properties of these flavonoids, we compared their effects on lipopolysaccharide induced release of inflammatory mediators from BV2 microglia. Both rhamnetin and sakuranetin reduced mediator release, but differed in potency (rhamnetin>sakuranetin) and the Hill slope of their concentration-response curves. For rhamnetin the Hill coefficient was >3.0 whereas for sakuranetin the coefficient was 1.0, suggesting that effects of rhamnetin are mediated through more than one mechanism, whereas sakuranetin has a single mechanism. nAChR antagonists decreased the Hill coefficient for rhamnetin toward unity, which suggests that a nAChR-mediated mechanism contributes cooperatively to its overall anti-inflammatory effect. In contrast nAChR antagonists had no effect on the potency or Hill coefficient for sakuranetin, but a concentration of nicotine (1?M) that had no effect alone, significantly increased the Hill coefficient of this flavonoid. In conclusion, the anti-inflammatory effects of rhamnetin benefit cooperatively from a nAChR-mediated mechanism. This action, together with potent free radical scavenging activity, suggests that flavonoids with alpha7 nAChR activity have therapeutic potential in neuroinflammatory conditions.
A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n?=?6x?=?42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.
Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.
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