Functional characterization of two 3-dehydroquinases of AroQ1 and AroQ2 in the shikimate pathway and expression of genes for the type III secretion system in Ralstonia solanacearum.

The shikimate pathway is a general route for the biosynthesis of aromatic amino acids (AAAs) in many microorganisms. A 3-dehydroquinase, AroQ, controls the third step of the shikimate pathway that catalyzes the formation of 3-dehydroquinate from 3-dehydroshikimate via a trans-dehydration reaction. Ralstonia solanacearum harbors two 3-dehydroquinases, AroQ1 and AroQ2, sharing 52% similarity in amino acids. Here, we demonstrated that two 3-dehydroquinases, AroQ1 and AroQ2, are essential for the shikimate pathway in R. solanacearum. The growth of R. solanacearum was completely diminished in a nutriment-limited medium with the deletion of both aroQ1 and aroQ2, while substantially impaired in planta. The aroQ1/2 double mutant was able to replicate in planta but grew slowly, which was ~4 orders of magnitude less than the parent strain to proliferate to the maximum cell densities in tomato xylem vessels. Moreover, the aroQ1/2 double mutant failed to cause disease in tomato and tobacco plants, whereas the deletion of either aroQ1 or aroQ2 did not alter the growth of R. solanacearum or pathogenicity on host plants. Supplementary shikimic acid (SA), an important intermediate of the shikimate pathway, substantially restored the diminished or impaired growth of aroQ1/2 double mutant in a limited medium or inside host plants. The necessity of AroQ1 and AroQ2 on the pathogenicity of solanacearum toward host plants was partially due to insufficient SA inside host plants. Moreover, the deletion of both aroQ1 and aroQ2 significantly impaired the expression of genes for the type III secretion system (T3SS) both in vitro and in planta. Its involvement in the T3SS was mediated through the well-characterized PrhA signaling cascade and was independent of growth deficiency under nutrient-limited conditions. Taken together, R. solanacearum 3-dehydroquinases play important roles in bacterial growth, the expression of the T3SS, and pathogenicity in host plants. These results could extend our insights into the understanding of the biological function of AroQ and the sophisticated regulation of the T3SS in R. solanacearum.

Evidence for distinct neuro-metabolic phenotypes in humans.

Advances in magnetic resonance imaging have shown how individual differences in the structure and function of the human brain relate to health and cognition. The relationship between individual differences and the levels of neuro-metabolites, however, remains largely unexplored - despite the potential for the discovery of novel behavioural and disease phenotypes. In this study, we measured 14 metabolite levels, normalised as ratios to total-creatine, with 1H magnetic resonance spectroscopy (MRS) acquired from the bilateral anterior cingulate cortices of six healthy participants, repeatedly over a period of four months. ANOVA tests revealed statistically significant differences of 3 metabolites and 3 commonly used combinations (total-choline, glutamate + glutamine and total-N-acetylaspartate) between the participants, with scyllo-inositol (F=85, p=6e-26) and total-choline (F=39, p=1e-17) having the greatest discriminatory power. This was not attributable to structural differences. When predicting individuals from the repeated MRS measurements, a leave-one-out classification accuracy of 88% was achieved using a support vector machine based on scyllo-inositol and total-choline levels. Accuracy increased to 98% with the addition of total-N-acetylaspartate and myo-inositol - demonstrating the efficacy of combining MRS with machine learning and metabolomic methodology. These results provide evidence for the existence of neuro-metabolic phenotypes, which may be non-invasively measured using widely available 3 Tesla MRS. Establishing these phenotypes in a larger cohort and investigating their connection to brain health and function presents an important area for future study.

GPR124 functions as a WNT7-specific coactivator of canonical ß-catenin signaling.

G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of ß-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of ß-catenin signaling in brain endothelium. WNT7-stimulated ß-catenin signaling was dependent upon GPR124's intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical ß-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barrier-genesis.