Bar coding MS2 spectra for metabolite identification

[Image: see text] Metabolite identifications are most frequently achieved in untargeted metabolomics by matching precursor mass and full, high-resolution MS(2) spectra to metabolite databases and standards. Here we considered an alternative approach for establishing metabolite identifications that does not rely on full, high-resolution MS(2) spectra. First, we select mass-to-charge regions containing the most informative metabolite fragments and designate them as bins. We then translate each metabolite fragmentation pattern into a binary code by assigning 1’s to bins containing fragments and 0’s to bins without fragments. With 20 bins, this binary-code system is capable of distinguishing 96% of the compounds in the METLIN MS(2) library. A major advantage of the approach is that it extends untargeted metabolomics to low-resolution triple quadrupole (QqQ) instruments, which are typically less expensive and more robust than other types of mass spectrometers. We demonstrate a method of acquiring MS(2) data in which the third quadrupole of a QqQ instrument cycles over 20 wide isolation windows (coinciding with the location and width of our bins) for each precursor mass selected by the first quadrupole. Operating the QqQ instrument in this mode yields diagnostic bar codes for each precursor mass that can be matched to the bar codes of metabolite standards. Furthermore, our data suggest that using low-resolution bar codes enables QqQ instruments to make MS(2)-based identifications in untargeted metabolomics with a specificity and sensitivity that is competitive to high-resolution time-of-flight technologies

MYC sensitises cells to apoptosis by driving energetic demand

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism

Evidence that 2-hydroxyglutarate is not readily metabolized in colorectal carcinoma cells

BACKGROUND: Two-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action. RESULTS: By applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly (13)C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in (13)C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from (13)C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period. CONCLUSIONS: Taken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products

Atmospheric circulation of tidally locked exoplanets: a suite of benchmark tests for dynamical solvers

The complexity of atmospheric modelling and its inherent non-linearity, together with the limited amount of data of exoplanets available, motivate model intercomparisons and benchmark tests. In the geophysical community, the Held-Suarez test is a standard benchmark for comparing dynamical core simulations of the Earth's atmosphere with different solvers, based on statistically-averaged flow quantities. In the present study, we perform analogues of the Held-Suarez test for tidally-locked exoplanets with the GFDL-Princeton Flexible Modeling System (FMS) by subjecting both the spectral and finite difference dynamical cores to a suite of tests, including the standard benchmark for Earth, a hypothetical tidally-locked Earth, a "shallow" hot Jupiter model and a "deep" model of HD 209458b. We find qualitative and quantitative agreement between the solvers for the Earth, tidally-locked Earth and shallow hot Jupiter benchmarks, but the agreement is less than satisfactory for the deep model of HD 209458b. Further investigation reveals that closer agreement may be attained by arbitrarily adjusting the values of the horizontal dissipation parameters in the two solvers, but it remains the case that the magnitude of the horizontal dissipation is not easily specified from first principles. Irrespective of radiative transfer or chemical composition considerations, our study points to limitations in our ability to accurately model hot Jupiter atmospheres with meteorological solvers at the level of ten percent for the temperature field and several tens of percent for the velocity field. Direct wind measurements should thus be particularly constraining for the models. Our suite of benchmark tests also provides a reference point for researchers wishing to adapt their codes to study the atmospheric circulation regimes of tidally-locked Earths/Neptunes/Jupiters.Comment: Accepted by MNRAS, 23 pages, 17 figures, 2 tables. No changes from previous version, except MNRAS wants no hyphen in the title. Sample movies of simulations are available at http://www.phys.ethz.ch/~kheng/fms

Energy Cost of Slow and Normal Gait Speeds in Low and Normally Functioning Adults

Objective Slow walking speed paired with increased energy cost is a strong predictor for mortality and disability in older adults but has yet to be examined in a heterogeneous sample (ie, age, sex, disease status). The aim of this study was to examine energy cost of slow and normal walking speeds among low- and normal-functioning adults. Design Adults aged 20–90 yrs were recruited for this study. Participants completed a 10-m functional walk test at a self-selected normal walking speed and were categorized as low functioning or normal functioning based on expected age- and sex-adjusted average gait speed. Participants completed two successive 3-min walking stages, at slower than normal and normal walking speeds, respectively. Gas exchange was measured and energy cost per meter (milliliter per kilogram per meter) was calculated for both walking speeds. Results Energy cost per meter was higher (P \u3c 0.0001) in the low-functioning group (n = 76; female = 59.21%; mean ± SD age = 61.13 ± 14.68 yrs) during the slower than normal and normal (P \u3c 0.0001) walking speed bouts compared with the normal-functioning group (n = 42; female = 54.76%; mean ± SD age = 51.55 ± 19.51 yrs). Conclusions Low-functioning adults rely on greater energy cost per meter of walking at slower and normal speeds. This has implications for total daily energy expenditure in low-functioning, adult populations

Attenuation of Experimental Aortic Aneurysm Formation in P-Selectin Knockout Mice

The aim of this study was to determine the role of P-selectin, an adhesion molecule found on the surface of activated platelets and endothelial cells during experimental aortic aneurysm formation. Infrarenal abdominal aortas of C57 black wild-type (WT) mice and P-selectin knockout (PKO) mice were measured in situ and then perfused with porcine pancreatic elastase (0.332 U mL). Whole blood was drawn from the tail artery on day 2 pre-perfusion to determine total and differential white blood cell (WBC) counts. On day 14 postperfusion, aortic diameters (AD) of WT mice ( N 19) and PKO mice ( N 9) were measured. An aortic aneurysm was defined as a 100 or greater increase in AD from pre-perfusion measurement. Immunohistochemistry, including H&E, trichrome and von Gieson staining, was performed on harvested aortic tissue. Statistical analysis was performed by t -test and Fisher's exact test. There were no significant differences in peripheral leukocyte counts at baseline between the two groups. WT mice had significantly larger AD compared to PKO mice at day 14 postperfusion (116 vs. 38 , P < 0.001). Aortic aneurysm penetrance was 52 in WT mice, while 0 ( P 0.01) of PKO mice formed aneurysms. On histologic examination, WT mouse aortas were associated with a significant inflammatory response and degradation of elastin and collagen fibers, while PKO mouse aortas lacked signs of inflammation or vessel wall injury. P-selectin deficiency attenuates aneurysm formation in the elastase aortic perfusion model. This was associated with a blunting of the inflammatory response and preserved vessel wall intergrity following elastase perfusion in the P-selectin knockout mice. Further investigation to elucidate the independent contributions of endothelial cell and platelet P-selectin in experimental aortic aneurysm formation is required.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73125/1/annals.1383.014.pd

Unexpected Inhibition of the Lipid Kinase PIKfyve Reveals an Epistatic Role for P38 MAPKs in Endolysosomal Fission and Volume Control

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission

Using mass spectrometry imaging to map fluxes quantitatively in the tumor ecosystem

Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, desorption electrospray ionization, and matrix assisted laser desorption ionization reveals alterations in multiple anabolic pathways. De novo fatty acid synthesis flux is increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux is elevated even higher at 8-fold relative to surrounding healthy tissue and highlights the importance of elongase activity in glioma

Optical modeling and polarization calibration for CMB measurements with ACTPol and Advanced ACTPol

The Atacama Cosmology Telescope Polarimeter (ACTPol) is a polarization sensitive upgrade to the Atacama Cosmology Telescope. Located at an elevation of 5190 m, ACTPol measures the Cosmic Microwave Background (CMB) temperature and polarization with arcminute-scale angular resolution. Calibration of the detector angles is a critical step in producing maps of the CMB polarization. Polarization angle offsets in the detector calibration can cause leakage in polarization from E to B modes and induce a spurious signal in the EB and TB cross correlations, which eliminates our ability to measure potential cosmological sources of EB and TB signals, such as cosmic birefringence. We present our optical modeling and measurements associated with calibrating the detector angles in ACTPol.Comment: 12 pages, 8 figures, conference proceedings submitted to Proceedings of SPIE; added reference in section 2 and merged repeated referenc