Benjamin F Cravatt

The Scripps Research Institute, La Jolla, California, United States

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Publications (423)3281.17 Total impact

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    ABSTRACT: Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.
    ACS Chemical Biology 12/2014; · 5.44 Impact Factor
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    ABSTRACT: Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell reports. 11/2014;
  • Benjamin F Cravatt
    ACS Chemical Neuroscience 11/2014; 5(11):1083. · 3.87 Impact Factor
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    ABSTRACT: A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle (intracranial self-stimulation, ICSS), which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), while peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide; AEA) and 2-AG. The CB1 receptor antagonist rimonabant, but not the CB2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and non-reinforced behaviors.
    Journal of Pharmacology and Experimental Therapeutics 11/2014; · 3.89 Impact Factor
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    ABSTRACT: Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol (2-AG), by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified, recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity - PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1-AG but not 2-AG or arachidonoylethanolamide (AEA). Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.
    The Journal of biological chemistry. 10/2014;
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    ABSTRACT: Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2(-/-) mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2(-/-) mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2(-/-) brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.
    Proceedings of the National Academy of Sciences of the United States of America. 09/2014;
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    ABSTRACT: Cannabinoid receptor agonists have been repeatedly shown to have anti-pyrogenic effects in a variety of assays. In rodents, high doses of CB1 cannabinoid receptor agonists induce hypothermia. Recently, attention has turned to how endocannabinoids (eCBs), including 2-arachidonoyglycerol (2-AG) and anandamide (AEA), regulate a host of homeostatic processes including thermoregulation. Exogenous administration of 2-AG or AEA has limited efficacy, because of their rapid degradation by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. Inhibiting either enzyme elevates levels of each respective target eCB. This experiment tested the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. Male C57BL/6 J mice were administered a MAGL or FAAH inhibitor and challenged with a cold ambient environment or the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip). MAGL inhibition exacerbated cold-induced hypothermia. Pretreatment with the selective CB1 receptor antagonist rimonabant blocked this decrease, whereas the selective CB2 antagonist SR144528 had no effect. MAGL inhibition also exacerbated LPS-induced hypothermia via CB1 receptors. In contrast, FAAH inhibition had no effect on Tb following either stressor. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses, neither MAGL nor FAAH inhibitors affect normal body temperature. However, MAGL inhibition leads to a dysregulation of Tb via a CB1 receptor mechanism of action in mice subjected to physical or physiological stress.
    Brain Behavior and Immunity 09/2014; 40:e11. · 5.61 Impact Factor
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    ABSTRACT: Background and PurposeAbrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide; AEA), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored.Experimental ApproachesTo evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor JZL184 and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets.Key ResultsBXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor JZL184 dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB1 receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans.Conclusion and ImplicationsOverall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.
    British Journal of Pharmacology 09/2014; · 5.07 Impact Factor
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    ABSTRACT: A graphical abstract is available for this content
    Chemical Society Reviews 08/2014; · 24.89 Impact Factor
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    ABSTRACT: To determine the role of the endocannabinoid, 2-arachodonyl glycerol (2-AG), in the regulation of nausea and vomiting.
    Psychopharmacology 08/2014; · 4.06 Impact Factor
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    ABSTRACT: The endocannabinoid ligand 2-arachidonoylglycerol (2-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine (BrdU)-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184. © 2014 Wiley Periodicals, Inc.
    Hippocampus 08/2014; · 5.49 Impact Factor
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    ABSTRACT: Phenotypic screening provides a means to discover small molecules that perturb cell biological processes. Discerning the proteins and biochemical pathways targeted by screening hits, however, remains technically challenging. We recently described the use of small molecules bearing photoreactive groups and latent affinity handles as fully functionalized probes for integrated phenotypic screening and target identification. The general utility of such probes, or, for that matter, any small-molecule screening library, depends on the scope of their protein interactions in cells, a parameter that remains largely unexplored. Here, we describe the synthesis of an ∼60-member fully functionalized probe library, prepared from Ugi-azide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively. In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function. For many of these proteins, ligands have not yet been described. Most of the probe-protein interactions showed well-defined structure-activity relationships across the probe library and were blocked by small-molecule competitors in cells. These findings indicate that fully functionalized small molecules canvas diverse segments of the human proteome and hold promise as pharmacological probes of cell biology.
    Journal of the American Chemical Society 07/2014; · 10.68 Impact Factor
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    ABSTRACT: Common pharmacological treatments of neuropathic and chronic inflammatory pain conditions generally lack efficacy and/or are associated with significant untoward side effects. However, recent preclinical data indicate that combined inhibition of cyclooxygenase (COX) and fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of the endocannabinoid N- arachidonoylethanolamine (anandamide; AEA), produces enhanced antinociceptive effects in a variety of murine models of pain. Accordingly, the primary objective of the present study was to investigate the consequences of co-administration of the COX inhibitor diclofenac and the highly selective FAAH inhibitor PF-3845 in models of neuropathic pain (i.e., chronic constrictive injury of the sciatic nerve (CCI)) and inflammatory pain induced by an intraplantar injection of carrageenan. Here, we report that combined administration of subthreshold doses of these drugs produced enhanced antinociceptive effects in CCI and carrageenan pain models, the latter of which was demonstrated to require both CB1 and CB2 receptors. The combined administration of subthreshold doses of these drugs also increased AEA levels and decreased prostaglandin levels in whole brain. Together, these data add to the growing research that dual blockade of FAAH and COX represents a potential therapeutic strategy for the treatment of neuropathic and inflammatory pain states.
    Pharmacology Biochemistry and Behavior 07/2014; · 2.82 Impact Factor
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    ABSTRACT: Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.
    Nature Communications 07/2014; 5:4421. · 10.74 Impact Factor
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    ABSTRACT: The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.
    Neuron 07/2014; 83(2):361-371. · 15.77 Impact Factor
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    ABSTRACT: Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.
    Nature Chemical Biology 07/2014; · 12.95 Impact Factor
  • Micah J Niphakis, Benjamin F Cravatt
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    ABSTRACT: Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.
    Annual review of biochemistry. 06/2014; 83:341-377.
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    ABSTRACT: Complementary genetic and pharmacological approaches to inhibit the monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), enable the exploration of potential therapeutic applications and physiological roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond inhibition of either enzyme alone. While CB1 receptor function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of high dose of the MAGL inhibitor, JZL184 (40 mg/kg) given acutely or for six days in FAAH (-/-) and (+/+) mice. While acute administration of JZL184 to FAAH (-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of THC, decreases in CB1 receptor agonist stimulated [(35)S]GTPγS binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity, but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.
    Journal of Pharmacology and Experimental Therapeutics 05/2014; · 3.89 Impact Factor
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    ABSTRACT: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from human pathogens S. aureus and P. aeruginosa can be readily inhibited by reactive oxygen species (ROS)-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 rapidly induces glycolysis and pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces metabolic levels in glycolysis and PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Both GapR and HexR may directly sense oxidative stress such as menadione.
    Journal of bacteriology 04/2014; · 3.94 Impact Factor

Publication Stats

21k Citations
3,281.17 Total Impact Points


  • 1994–2014
    • The Scripps Research Institute
      • • Department of Chemical Physiology
      • • Skaggs Institute for Chemical Biology
      • • Department of Chemistry
      • • Department of Cell and Molecular Biology
      La Jolla, California, United States
  • 2013
    • Abide Therapeutics
      San Diego, California, United States
    • Liverpool School of Tropical Medicine
      Liverpool, England, United Kingdom
  • 2011–2013
    • West Virginia University
      • Department of Psychology
      Morgantown, West Virginia, United States
  • 2009–2013
    • Max Planck Institute for Plant Breeding Research
      Köln, North Rhine-Westphalia, Germany
  • 2005–2013
    • National Institutes of Health
      • • Laboratory of Physiologic Studies
      • • Section on Neuroendocrinology
      Bethesda, MD, United States
  • 2002–2012
    • Virginia Commonwealth University
      • Department of Pharmacology and Toxicology
      Richmond, VA, United States
  • 2009–2011
    • Medical College of Wisconsin
      • • Cell Biology, Neurobiology and Anatomy
      • • Department of Pharmacology and Toxicology
      Milwaukee, WI, United States
  • 2007–2011
    • Pfizer Inc.
      • Pfizer Global Research & Development
      New York City, New York, United States
    • University of Rome Tor Vergata
      • • Dipartimento di Medicina dei Sistemi
      • • Dipartimento di Biologia
      Roma, Latium, Italy
  • 2010
    • University of Washington Seattle
      Seattle, Washington, United States
  • 2008–2009
    • Indiana University Bloomington
      • Department of Psychological and Brain Sciences
      Bloomington, IN, United States
    • Università degli Studi di Teramo
      • Department of Comparative Biomedical Sciences
      Teramo, Abruzzo, Italy
  • 2006–2008
    • Vanderbilt University
      • • Center in Molecular Toxicology
      • • Department of Biochemistry
      • • Department of Pediatrics
      Nashville, MI, United States
  • 2005–2008
    • University of California, Berkeley
      • Department of Environmental Science, Policy, and Management
      Berkeley, MO, United States
  • 2003
    • National Institute on Alcohol Abuse and Alcoholism
      Maryland, United States
  • 1998–2003
    • Queen Mary, University of London
      Londinium, England, United Kingdom
  • 2000
    • University of London
      • School of Biological Sciences
      Londinium, England, United Kingdom