Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut ''microbiome'' on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas Ϸ10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.

Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.

Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

Cancer cells need to meet the metabolic demands of rapid cell growth within a continually changing microenvironment. Genetic mechanisms for reprogramming cellular metabolism toward proliferative, pro-survival pathways are well-reported. However, post-translational mechanisms, which would enable more rapid, reversible adaptations of cellular metabolism in response to protein signaling or environmental sensing systems, are less well understood. Here we demonstrate that the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a key metabolic regulator of glucose metabolism. O-GlcNAc is dynamically induced at Ser529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibits PFK1 activity and redirects the flux of glucose from glycolysis through the pentose phosphate pathway (PPP), thereby conferring a selective growth advantage to cancer cells. Blocking glycosylation of PFK1 at Ser529 reduced cancer cell proliferation in vitro and impaired tumor formation in vivo. These studies reveal an unexpected mechanism for the regulation of metabolic enzymes and pathways, and pinpoint a new therapeutic approach for combating cancer.

Monolithic columns for capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propane-sulfonic acid (AMPS) in the presence of a porogenic solvent. The use of these novel macroporous monoliths eliminates the need for frits, the difficulties encountered with packed capillaries, and capillary surface functionalization. Since the porous properties of the monolithic materials can be easily tailored through changes in the composition of the ternary porogenic solvent, the effects of both pore size and the percentage of sulfonic acid monomer on the efficiency and the electroosmotic flow velocity of the capillary columns could be studied independently over a broad range. A simple increase in the content of charged functionalities within the monolith leads to an expected acceleration of the flow velocity. However, increasing the pore size leads to a substantial deterioration of the efficiency of the separation. In contrast, monoliths with increasing levels of AMPS in which the pore size remains fixed due to adjustments in the composition of the porogenic solvent show no deterioration in efficiency while maintaining the same increase in flow velocity, thus producing a significant reduction in separation time. Additionally, measurements on monoliths with constant levels of AMPS but different pore sizes suggest that flow velocity may be affected by the flow resistance within the capillary column.