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Background Neonatal abstinence syndrome is an array of signs and symptoms experienced by a newborn due to abrupt discontinuation of intrauterine exposure to certain drugs, primarily opioids. In the United States, the incidence of neonatal abstinence syndrome has tripled over the past decade. The current standard of care for drug testing includes the analysis of infant urine and meconium. Sample collection is associated with several limitations, including diaper media interferences, limited sample amount, sample heterogeneity, and the need for professional staff for collection. Umbilical cord tissue has emerged as a convenient sample matrix for testing owing to its universal availability. The purpose of this study was to examine umbilical cords using an untargeted metabolomics approach to determine the detected drugs and validate an analytical method to confirm and quantify the identified drugs. Methods A metabolomics analysis was performed with 21 umbilical cords to screen for drugs and drug metabolites by liquid chromatography-mass spectrometry. Drugs were identified using the National Institute of Standards and Technology database, and an analytical method was developed and validated using secondary liquid chromatography-mass spectrometry instrument for positive confirmation and quantitative analysis. Results Twenty-one random umbilical cords from women were tested: 4 were positive for cocaine and the primary and secondary metabolites; one was positive for methadone, the primary metabolite; 3 were positive for cotinine, the metabolite of nicotine; and 5 were positive for acetyl norfentanyl. Conclusions Our research is a prospective method development study using untargeted and targeted approaches to characterize steady-state drug metabolite levels in the umbilical cord matrix at the time of delivery. By characterizing drug type and concentration, this methodology can be used to develop a reliable complementary testing method for meconium toxicology screens.

Currently there is great interest in developing cytotoxic pharmacotherapies that disrupt mitochondrial energy transduction in cancer. Given that mitochondria are critical to mammalian energy homeostasis, clinical success of a given therapeutic will undoubtedly hinge upon its cancer cell selectivity. That said, how the mitochondrial network is intrinsically remodeled to drive/enable the cancer phenotype remains a biological black box, largely due to limitations in analytical approaches. Herein, we leveraged an in-house diagnostic biochemical workflow to comprehensively evaluate mitochondrial bioenergetic efficiency and capacity in human leukemia. Using this platform, we provide direct evidence that despite minimal changes in absolute respiratory kinetics, leukemic mitochondria are hallmarked by intrinsic limitations in oxidative phosphorylation (OXPHOS) that constrain the network’s ability to contribute to cellular ATP free energy (i.e, ΔGATP) charge. Together, these findings link accelerated oxidative metabolism in leukemia to intrinsic OXPHOS deficiency and provide proof-of-concept that restoring, rather than disrupting, OXPHOS across the leukemic mitochondrial network may represent an untapped, but highly feasible, therapeutic avenue.

Lipases, a versatile class of biocatalysts, have been shown to function in non-aqueous media/organic solvents and to possess &ldquo, promiscuous&rdquo, catalytic activity for a wide range of organic transformations. In this study, we explored the biocatalytic properties of a library of commercially available lipases by screening them for catalysis of a one-pot synthesis of Wieland&ndash, Miescher ketone, an important intermediate in the synthesis of biologically active compounds such as steroids and terpenoids, from methyl vinyl ketone and 2-methyl-1,3-cyclohexanedione. As a direct outgrowth of this screen, we created an optimized procedure for Wieland&ndash, Miescher ketone (WMK) synthesis using crude lipase preparations, characterizing both reaction yield and enantiomeric excess. We also identified principal components of the crude lipase mixture through proteomics and present evidence for a non-lipolytic origin of the observed catalysis. Finally, using the optimized conditions developed in this study, we propose a general absorbance-based screening methodology for assessing biocatalytic potential of crude enzyme preparations for synthesis of WMK.

Lipases, a versatile class of biocatalysts, have been shown to function in non-aqueous media/organic solvents and to possess promiscuous catalytic activity for a wide range of organic transformations. In this study, we explore the biocatalytic properties of a library of commercially available lipases by screening them for catalysis of a one-pot synthesis of Wieland-Miescher Ketone, an important intermediate in the synthesis of biologically active compounds such as steroids and terpenoids, from methyl vinyl ketone and 2-methyl-1,3-cyclohexanedione. As a direct outgrowth of this screen, we have created an optimized procedure for Wieland-Miescher Ketone (WMK) synthesis using crude lipase preparations, characterizing both reaction yield and enantiomeric excess. We have also identified principal components of the crude lipase mixture through proteomics and present evidence for a non-lipolytic origin of the observed catalysis. Finally, using the optimized conditions developed in this study, we propose a general absorbance-based screening methodology for assessing biocatalytic potential of crude enzyme preparations for synthesis of WMK.

Barth Syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy and muscle weakness. The underlying cause of BTHS is a mutation in the tafazzin (TAZ) gene, a key enzyme of cardiolipin biosynthesis. The lack of CL arising from loss of TAZ function results in destabilization of the electron transport system, promoting oxidative stress that is thought to contribute to development of cardioskeletal myopathy. Indeed, in vitro studies demonstrate that mitochondria-targeted antioxidants improve contractile capacity in TAZ-deficient cardiomyocytes. The purpose of the present study was to determine if resolving mitochondrial oxidative stress would be sufficient to prevent cardiomyopathy and skeletal myopathy in vivo using a mouse model of BTHS. To this end we crossed mice that overexpress catalase in the mitochondria (MCAT mice) with TAZ-deficient mice (TAZKD) to produce TAZKD mice that selectively overexpress catalase in the mitochondria (TAZKD+MCAT mice). TAZKD+MCAT mice exhibited decreased mitochondrial H(2)O(2) emission and lipid peroxidation compared to TAZKD littermates, indicating decreased oxidative stress. Despite the improvements in oxidative stress, TAZKD+MCAT mice developed cardiomyopathy and mild muscle weakness similar to TAZKD littermates. These findings indicate that resolving oxidative stress is not sufficient to suppress cardioskeletal myopathy associated with BTHS.

EphrinA1 is a tyrosine kinase receptor localized in the cellular membrane of healthy cardiomyocytes, the expression of which is lost upon myocardial infarction (MI). Intra-cardiac injection of the recombinant form of ephrinA1 (ephrinA1-Fc) at the time of ligation in mice has shown beneficial effects by reducing infarct size and myocardial necrosis post-MI. To date, immunohistochemistry and Western blotting comprise the only experimental approaches utilized to localize and quantify relative changes of ephrinA1 in sections and homogenates of whole left ventricle, respectively. Herein, we used matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) coupled with a time-of-flight mass spectrometer (MALDI/TOF MS) to identify intact as well as tryptic fragments of ephrinA1 in healthy controls and acutely infarcted murine hearts. The purpose of the present study was 3-fold: (1) to spatially resolve the molecular distribution of endogenous ephrinA1, (2) to determine the anatomical expression profile of endogenous ephrinA1 after acute MI, and (3) to identify molecular targets of ephrinA1-Fc action post-MI. The tryptic fragments detected were identified as the ephrinA1-isoform with 38% and 34% sequence coverage and Mascot scores of 25 for the control and MI hearts, respectively. By using MALDI-MSI, we have been able to simultaneously measure the distribution and spatial localization of ephrinA1, as well as additional cardiac proteins, thus offering valuable information for the elucidation of molecular partners, mediators, and targets of ephrinA1 action in cardiac muscle. Graphical Abstractᅟ Electronic supplementary material The online version of this article (10.1007/s13361-017-1869-7) contains supplementary material, which is available to authorized users.

Objective Phosphatidylethanolamine methyltransferase (PEMT) generates phosphatidylcholine (PC), the most abundant phospholipid in the mitochondria and an important acyl chain donor for cardiolipin (CL) biosynthesis. Mice lacking PEMT (PEMTKO) are cold-intolerant when fed a high-fat diet (HFD) due to unclear mechanisms. The purpose of this study was to determine whether PEMT-derived phospholipids are important for the function of uncoupling protein 1 (UCP1) and thus for maintenance of core temperature. Methods To test whether PEMT-derived phospholipids are important for UCP1 function, we examined cold-tolerance and brown adipose (BAT) mitochondria from PEMTKO mice with or without HFD feeding. We complemented these studies with experiments on mice lacking functional CL due to tafazzin knockdown (TAZKD). We generated several conditional mouse models to study the tissue-specific roles of PEMT, including mice with BAT-specific knockout of PEMT (PEMT-BKO). Results Chow- and HFD-fed PEMTKO mice completely lacked UCP1 protein in BAT, despite a lack of difference in mRNA levels, and the mice were accordingly cold-intolerant. While HFD-fed PEMTKO mice exhibited reduced mitochondrial CL content, this was not observed in chow-fed PEMTKO mice or TAZKD mice, indicating that the lack of UCP1 was not attributable to CL deficiency. Surprisingly, the PEMT-BKO mice exhibited normal UCP1 protein levels. Knockout of PEMT in the adipose tissue (PEMT-AKO), liver (PEMT-LKO), or skeletal muscle (PEMT-MKO) also did not affect UCP1 protein levels, suggesting that lack of PEMT in other non-UCP1-expressing cells communicates to BAT to suppress UCP1. Instead, we identified an untranslated UCP1 splice variant that was triggered during the perinatal period in the PEMTKO mice. Conclusions PEMT is required for UCP1 splicing that yields functional protein. This effect is derived by PEMT in nonadipocytes that communicates to BAT during embryonic development. Future research will focus on identifying the non-cell-autonomous PEMT-dependent mechanism of UCP1 splicing., Highlights • Whole-body ablation of PEMT results in a complete loss of UCP1 protein. • While PEMT-derived PC may participate in CL biosynthesis, changes in mitochondrial PC or CL do not explain the loss of UCP1 protein. • PEMT regulates brown adipose UCP1 in a non-cell-autonomous manner. • PEMT deletion promotes the use of an alternative 3’ splice junction in exon 5 of UCP1, preventing UCP1 protein expression.

A commercial liquid chromatography/drift tube ion mobility-mass spectrometer (LC/IM-MS) was evaluated for its utility in global metabolomics analysis. Performance was assessed using 12 targeted metabolite standards where the limit of detection (LOD), linear dynamic range, resolving power, and collision cross section (Ω) are reported for each standard. Data were collected in three different instrument operation modes: flow injection analysis with IM-MS (FIA/IM-MS), LC/MS, and LC/IM-MS. Metabolomics analyses of human plasma and HaCaT cells were used to compare the above three operation modes. LC/MS provides linearity in response, data processing automation, improved limits of detection, and ease of use. Advantages of LC/IM-MS and FIA/IM-MS include the ability to develop mobility-mass trend lines for structurally similar biomolecules, increased peak capacity, reduction of chemical/matrix noise, improvement in signal-to-noise, and separations of isobar/isomer compounds that are not resolved by LC. We further tested the feasibility of incorporating IM-MS into conventional LC/MS metabolomics workflows. In general, the addition of ion mobility dimension has increased the separation of compounds in complex biological matrixes and has the potential to largely improve the throughput of metabolomics analysis.