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1.  Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells 
PLoS ONE  2015;10(6):e0129029.
AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.
PMCID: PMC4480354  PMID: 26107620
2.  Untargeted LC-MS Metabolomics of Bronchoalveolar Lavage Fluid Differentiates Acute Respiratory Distress Syndrome from Health 
Journal of proteome research  2013;13(2):640-649.
Acute respiratory distress syndrome (ARDS) remains a significant hazard to human health and is clinically challenging because there are no prognostic biomarkers and no effective pharmacotherapy. The lung compartment metabolome may detail the status of the local environment that could be useful in ARDS biomarker discovery and the identification of drug target opportunities. However, neither the utility of bronchoalveolar lavage fluid (BALF) as a biofluid for metabolomics nor the optimal analytical platform for metabolite identification are established. To address this, we undertook a study to compare metabolites in BALF samples from patients with ARDS and healthy controls using a newly developed liquid chromatography (LC)-mass spectroscopy (MS) platform for untargeted metabolomics. Following initial testing of three different high performance liquid chromatography (HPLC) columns, we determined that reversed phase (RP)-LC and hydrophilic interaction chromatography (HILIC), were the most informative chromatographic methods because they yielded the most and highest quality data. Following confirmation of metabolite identification, statistical analysis resulted in 37 differentiating metabolites in the BALF of ARDS compared with health across both analytical platforms. Pathway analysis revealed networks associated with amino acid metabolism, glycolysis and gluconeogenesis, fatty acid biosynthesis, phospholipids and purine metabolism in the ARDS BALF. The complementary analytical platforms of RPLC and HILIC-LC generated informative, insightful metabolomics data of the ARDS lung environment.
PMCID: PMC4068805  PMID: 24289193
biomarkers; critical illness; metabolomics; lung injury; bioinformatics; phospholipids; lactate; xanthine oxidase; hippurate; pharmacotherapy
3.  Impact of Anesthesia and Euthanasia on Metabolomics of Mammalian Tissues: Studies in a C57BL/6J Mouse Model 
PLoS ONE  2015;10(2):e0117232.
A critical application of metabolomics is the evaluation of tissues, which are often the primary sites of metabolic dysregulation in disease. Laboratory rodents have been widely used for metabolomics studies involving tissues due to their facile handing, genetic manipulability and similarity to most aspects of human metabolism. However, the necessary step of administration of anesthesia in preparation for tissue sampling is not often given careful consideration, in spite of its potential for causing alterations in the metabolome. We examined, for the first time using untargeted and targeted metabolomics, the effect of several commonly used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed dramatic, tissue-specific impacts of tissue collection strategy. Among many differences observed, post-euthanasia samples showed elevated levels of glucose 6-phosphate and other glycolytic intermediates in skeletal muscle. In heart and liver, multiple nucleotide and purine degradation metabolites accumulated in tissues of euthanized compared to anesthetized animals. Adipose tissue was comparatively less affected by collection strategy, although accumulation of lactate and succinate in euthanized animals was observed in all tissues. Among methods of tissue collection performed pre-euthanasia, ketamine showed more variability compared to isoflurane and pentobarbital. Isoflurane induced elevated liver aspartate but allowed more rapid initiation of tissue collection. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.
PMCID: PMC4319778  PMID: 25658945
4.  Splenic abscess rupture postappendicectomy 
BMJ Case Reports  2012;2012:bcr0120125705.
The authors present a case of splenic abscess rupture postappendicectomy. Splenic abscess is rare with a reported incidence of 0.05%–0.7%. It is extremely unusual for a splenic abscess to result in splenic rupture. Contiguous spread, in this case from postappendix perforation, can cause splenic abscess formation. Postemergency splenectomy, the patient required admission to intensive therapy unit for 5 days but made a good postoperative recovery. This case is important to report as this is a rare postoperative complication of generalised peritonitis and this case highlights that astute diagnosis and management of the deteriorating surgical patient and rapid mobilisation of theatre are lifesaving.
PMCID: PMC3416992  PMID: 22802559
5.  Alterations in Lipid Signaling Underlie Lipodystrophy Secondary to AGPAT2 Mutations 
Diabetes  2012;61(11):2922-2931.
Congenital generalized lipodystrophy (CGL), secondary to AGPAT2 mutation is characterized by the absence of adipocytes and development of severe insulin resistance. In the current study, we investigated the adipogenic defect associated with AGPAT2 mutations. Adipogenesis was studied in muscle-derived multipotent cells (MDMCs) isolated from vastus lateralis biopsies obtained from controls and subjects harboring AGPAT2 mutations and in 3T3-L1 preadipocytes after knockdown or overexpression of AGPAT2. We demonstrate an adipogenic defect using MDMCs from control and CGL human subjects with mutated AGPAT2. This defect was rescued in CGL MDMCs with a retrovirus expressing AGPAT2. Both CGL-derived MDMCs and 3T3-L1 cells with knockdown of AGPAT2 demonstrated an increase in cell death after induction of adipogenesis. Lack of AGPAT2 activity reduces Akt activation, and overexpression of constitutively active Akt can partially restore lipogenesis. AGPAT2 modulated the levels of phosphatidic acid, lysophosphatidic acid, phosphatidylinositol species, as well as the peroxisome proliferator–activated receptor γ (PPARγ) inhibitor cyclic phosphatidic acid. The PPARγ agonist pioglitazone partially rescued the adipogenic defect in CGL cells. We conclude that AGPAT2 regulates adipogenesis through the modulation of the lipome, altering normal activation of phosphatidylinositol 3-kinase (PI3K)/Akt and PPARγ pathways in the early stages of adipogenesis.
PMCID: PMC3478532  PMID: 22872237
6.  The Sedoheptulose Kinase CARKL Directs Macrophage Polarization through Control of Glucose Metabolism 
Cell Metabolism  2012;15(6):813-826.
Immune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.
Graphical Abstract
► Screened 199 human kinases for their immunoregulatory potential ► CARKL bridges glycolysis, the pentose phosphate pathway, and immune function ► CARKL focuses cellular metabolism toward a “high-redox” state ► CARKL regulation is required for macrophage polarization
PMCID: PMC3370649  PMID: 22682222
7.  Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production 
PLoS ONE  2011;6(5):e19710.
NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins.
PMCID: PMC3092764  PMID: 21589930
8.  Improved Statistical Methods are Needed to Advance Personalized Medicine 
Common methods of statistical analysis, e.g. Analysis of Variance and Discriminant Analysis, are not necessarily optimal in selecting therapy for an individual patient. These methods rely on group differences to identify markers for disease or successful interventions and ignore sub-group differences when the number of sub-groups is large. In these circumstances, they provide the same advice to an individual as the average patient. Personalized medicine needs new statistical methods that allow treatment efficacy to be tailored to a specific patient, based on a large number of patient characteristics. One such approach is the sequential k-nearest neighbor analysis (patients-like-me algorithm). In this approach, the k most similar patients are examined sequentially until a statistically significant conclusion about the efficacy of treatment for the patient-at-hand can be arrived at. For some patients, the algorithm stops before the entire set of data is examined and provides beneficial advice that may contradict recommendations made to the average patient. Many problems remain in creating statistical tools that can help individual patients but this is an important area in which progress in statistical thinking is helpful.
PMCID: PMC2911789  PMID: 20676226
K-nearest neighbor analysis; sequential analysis; personalized medicine; patients-like-me algorithm; statistical methods
9.  NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity 
BMC Chemical Biology  2010;10:2.
NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.
Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.
The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.
PMCID: PMC2834649  PMID: 20175898
A deoxyribonucleic acid preparation which showed infectivity and tumorigenic activity in domestic rabbits was isolated from the papillomatous tissue of wild cottontail rabbits by phenolic deproteinization procedure. The activity of the preparation could be completely abolished by its exposure to a minute amount (0.02 µg/ml) of DNAase. Antisera against Shope papilloma virus did not block the tumorigenic activity of the preparation, and trypsin and chymotrypsin had no effect on it. The extraction with phenol of a partially purified virus preparation also yielded extracts with tumorigenic potency. Extracts obtained from the domestic rabbit papilloma and submitted to phenolic deproteinization also proved infective and tumorigenic in rabbits of this sort, although the level of "tumorigenicity" was much lower than that of the cottontail preparations. Tests for intact virus, carried out with half of the extracts yielded wholly negative findings.
PMCID: PMC2180365  PMID: 19867197
1. The severity of herpetic infection, of mice varied according to the site of inoculation, decreasing in the following order: cornea, skin of abdomen, pad of hind foot, tail. 2. The preliminary treatment of the foot pad or tail with methylcholanthrene increased the susceptibility to herpetic infection to a limited extent. 3. Two-week-old mice showed a much greater susceptibility to herpes virus inoculated on the tail or on the pad of the hind foot than did adult mice. 4. The HF strain of herpes virus, after more than 125 serial brain-to-brain passages in mice, possessed high virulence for cutaneous tissues of mice and showed no important differences in this respect from a recently isolated strain (Klee) of herpes virus.
PMCID: PMC2135977  PMID: 15415507
Two-week-old mice inoculated with herpes virus on the pad of a hind foot regularly developed paralysis of the infected limb followed by paraplegia and encephalitis terminating fatally 5 or 6 days after inoculation. Hyperimmune rabbit serum given intraperitoneally at the time virus was inoculated on the foot pad prevented the formation of an herpetic lesion of the foot pad. When the antiserum was given 12 hours after inoculation of the virus, a typical infection of the epithelium of the foot pad developed, but the virus was prevented from causing obvious signs of infection of the nervous system in many of the animals. Amputation of the foot 2 hours after the inoculation of the virus prevented the paralysis of the hind leg. Some of the mice died of a delayed encephalitis. Amputation of the foot at 24 hours neither prevented nor delayed the sequence of paralysis of the hind leg, encephalitis, and death. In order to study immune serum therapy of an infection of the nervous system uncomplicated by a peripheral focus of infection or by traumatic disturbance of the central nervous system, 2-week-old mice were inoculated on the foot pad, the infected feet were amputated 24 hours later, and the immune serum was administered at varying intervals thereafter. Using litter mate controls and statistically significant numbers of mice, it was shown that hyperimmune rabbit serum, administered during the first one-third of the incubation period, retards and, in some cases, arrests the progress of herpetic infection within the nervous system.
PMCID: PMC2135659  PMID: 19871580
15.  Women in Industry* 
California and Western Medicine  1943;59(2):119-121.
PMCID: PMC1780403  PMID: 18746584

Results 1-16 (16)