Competing interests C.J.D. is on the Scientific Advisory Board of Mirati Therapeutics. A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS-regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.C.K. is on the Scientific Advisory Board of Cornerstone/Rafael Pharmaceuticals.

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and 1H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.

In-cell NMR 1-3 provides information about how the crowded environment in cells, where the concentration of macromolecules reaches hundreds of grams per liter, 4 affects protein structure and dynamics. Several successes, including target protein overexpression in Escherichia coli 1,5-9 and injection of isotope-enriched protein into Xenopus laevis oocytes, 10,11 have been reported, but in-cell NMR remains in its infancy, and several potential problems need to be addressed. One problem is protein leakage from the cell during the experiment. 12-14 When this occurs, sharp signals from the protein molecules in the less viscous media mask the broader signals from the protein molecules in the more viscous cytosol. Here we examine two proteins. The intrinsically disordered protein, α-synuclein (αSN, ∼14 kDa), does not leak and is observed by in-cell NMR. The globular protein, chymotrypsin inhibitor 2 (CI2, ∼7 kDa), 15 leaks, and the remaining intracellular CI2 is not detectable. We show that the difference in detectability between αSN and CI2 is consistent with a differential dynamical response to macromolecular crowding. Figure 1A shows the 15 N-1 H HSQC spectrum of an in-cell NMR experiment on αSN. The spectrum is consistent with that from previous studies. 9,16 Figure 1B shows the spectrum from the supernatant collected immediately after sample preparation. Only metabolite signals 17 are observed. Figure 1C shows the spectrum from the supernatant recovered after the in-cell NMR experiment. Again, only metabolites are observed. The data demonstrate that the αSN spectrum in panel A comes from αSN in the cell. We have obtained similar results with the intrinsically disordered protein FlgM. 8 We performed the same experiments with CI2 expressing cells. In contrast to αSN, all three spectra are nearly identical ( Figure 1E-G) (and typical of a CI2 spectrum 18 in dilute solution). These data suggest that CI2 leaks from the cells. SDS-PAGE confirms that ∼20% of the CI2 is lost from cells. Figure 1D), proving that encapsulated cells can provide useful in-cell spectra. NIH Public AccessWe repeated the experiment with CI2-expressing cells. No CI2 signal was observed even though we increased the sensitivity by accumulating the data for a longer time compared to the other samples ( Figure 1H). However, a typical CI2 spectrum was recovered after dissolving the encapsulates with EDTA (data not shown). These observations suggest that the signal from the intracellular CI2, which we know is present in detectable amounts, is too broad to observe. We reasoned that the broadening arises from an alteration in the dynamics of CI2, either from binding a larger species in cells or from the higher viscosity of E. coli cytoplasm, which can be 10-11 times that of water. 21,22Why would the intrinsically disordered proteins αSN and FlgM react differently compared with a globular protein CI2 to the increased viscosity in cells such that we detect αSN and FlgM, but not CI2? The ability to detect a protein by high-resolution NMR depends on its dy...

Methods for the simultaneous polarization of multiple 13 C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13 C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13 C sodium bicarbonate and [1-13 C]pyruvate in the same sample was achieved, resulting in high solution state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T 1 ) (46.7s and 47.7s respectively at 3T). Consistent with chemical shift anisotropy dominating the T 1 relaxation of carbonyls, T 1 values for 13 C bicarbonate and [1-13 C]pyruvate were even longer at 3T (49.7s and 67.3s, respectively). Co-polarized 13 C bicarbonate and [1-13 C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13 C sodium bicarbonate with 13 C CO 2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-13 C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13 C bicarbonate and [1-13 C] pyruvate or polarized [1-13 C]pyruvate alone. The technique was extended to polarize four 13 C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-13 C]pyruvic acid, 13 C sodium bicarbonate, [1,4-13 C]fumaric acid, and 13 C urea with high levels of solution polarization (17.5, 10.3, 15.6 and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Achieving the ability to identify individuals who are susceptible to drug-induced liver injury (DILI) would represent a major advance in personalized medicine. Clayton et al. demonstrated that the pattern of endogenous metabolites in urine could predict susceptibility to acetaminophen-induced liver injury in rats. We designed a clinical study to test this approach in healthy adults who received 4 g of acetaminophen per day for 7 days. Urine metabolite profiles obtained before the start of treatment were not sufficient to distinguish which of the subjects would develop mild liver injury, as indicated by a rise in alanine aminotransferase (ALT) to a level more than twice the baseline value (responders). However, profiles obtained shortly after the start of treatment, but prior to ALT elevation, could distinguish responders from nonresponders. Statistical analyses revealed that predictive metabolites included those derived from the toxic metabolite N-acetyl paraquinone imine (NAPQI), but that the inclusion of endogenous metabolites was required for significant prediction. This "early-intervention pharmaco-metabonomics" approach should now be tested in clinical trials of other potentially hepatotoxic drugs.