The feel of inclusivity

Collaboration between diverse groups with different perspectives promotes scientific discovery. To enhance these partnerships and facilitate communication, innovative advances in universal design are essential

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

Mitochondrial pyruvate metabolism and glutaminolysis toggle steady-state and emergency myelopoiesis

To define the metabolic requirements of hematopoiesis, we examined blood lineages in mice conditionally deficient in genes required for long-chain fatty acid oxidation (Cpt2), glutaminolysis (Gls), or mitochondrial pyruvate import (Mpc2). Genetic ablation of Cpt2 or Gls minimally impacted most blood lineages. In contrast, deletion of Mpc2 led to a sharp decline in mature myeloid cells and a slower reduction in T cells, whereas other hematopoietic lineages were unaffected. Yet MPC2-deficient monocytes and neutrophils rapidly recovered due to a transient and specific increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was progenitor intrinsic and accompanied by a metabolic switch to glutaminolysis. Myeloid recovery after loss of MPC2 or cyclophosphamide treatment was delayed in the absence of GLS. Reciprocally, MPC2 was not required for myeloid recovery after cyclophosphamide treatment. Thus, mitochondrial pyruvate metabolism maintains myelopoiesis under steady-state conditions, while glutaminolysis in progenitors promotes emergency myelopoiesis

A multidimensional metabolomics workflow to image biodistribution and evaluate pharmacodynamics in adult zebrafish

An integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper

Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation

Proliferating cells often have increased glucose consumption and lactate excretion relative to the same cells in the quiescent state, a phenomenon known as the Warburg effect. Despite an increase in glycolysis, however, here we show that non-transformed mouse fibroblasts also increase oxidative phosphorylation (OXPHOS) by nearly two-fold and mitochondrial coupling efficiency by ~30% during proliferation. Both increases are supported by mitochondrial fusion. Impairing mitochondrial fusion by knocking down mitofusion-2 (Mfn2) was sufficient to attenuate proliferation, while overexpressing Mfn2 increased proliferation. Interestingly, impairing mitochondrial fusion decreased OXPHOS but did not deplete ATP levels. Instead, inhibition caused cells to transition from excreting aspartate to consuming it. Transforming fibroblasts with th

Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging

BACKGROUND: Tissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3(′)-deoxy-3(′)-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses. METHODS: NIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt’s lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy. RESULTS: NIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism. CONCLUSIONS: NIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and provides new spatial and functional dimensions to studies of cancer metabolism without the need for radiotracers or tissue extraction. These findings should prove useful for in vitro and pre-clinical studies of cancer metabolism, and aid the optimization of metabolism-based cancer therapies and diagnostics

Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome

While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis