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Cocaine and methamphetamine remain highly abused drugs in the United States due to their euphoric effects. This study examines classical stimulant casework, defined as cases positive for methamphetamine and/or cocaine, received by the Toxicology Laboratory and the Drug Analysis Laboratory at the Dallas County Southwestern Institute of Forensic Sciences from local law enforcement agencies and/or the Office of the Medical Examiner (OME) between January 1, 2017, and December 31, 2022. Methamphetamine positivity increased from 10.4% to 20.3% in the Toxicology Laboratory over the 6 years, whereas cocaine positivity remained relatively stable at approximately 17%. Similarly, in the Drug Analysis Laboratory, the methamphetamine positivity rate changed from 24.8% to 33.2%, whereas cocaine identification remained stable at approximately 20%. Blood concentrations of methamphetamine in OME cases ranged from 10.1-42,740.0 ng/mL while they were lower in DWI casework ranging from 10.2-2385.0 ng/mL. The blood concentration trends of cocaine were similar to methamphetamine, with OME casework ranging higher (10.0-24,501.0 ng/mL) than DWI casework (10.2-371.6 ng/mL). Polydrug use was evident for both methamphetamine and cocaine in postmortem cases, and the top three most frequently co-occurring drug/drug class were opioids/opiates, cannabinoids, and ethanol. The results from this study aid in the understanding of historical usage trends of cocaine and methamphetamine in Dallas County and how those trends have changed over time as newer stimulant drugs have emerged., (© 2023 American Academy of Forensic Sciences.)

Alcohol (ethanol) is the most widely detected drug in forensic toxicology casework and an increase in consumption of alcohol was reported during the COVID-19 pandemic. The increase in consumption could be attributed to rising stress levels and social isolation. To determine whether the pandemic had an impact on ethanol positivity and concentrations in cases analyzed by the Dallas County Southwestern Institute of Forensic Sciences, blood ethanol results were evaluated from January 1, 2019, through December 31, 2021. This time frame captured ethanol prevalence and concentrations before, during, and immediately following the pandemic for comparison. The average ethanol concentration in postmortem casework over the three years for each quarter ranged from 0.116 g/100 mL to 0.142 g/100 mL while the average concentration in driving while intoxicated (DWI) was higher, ranging from 0.173 g/100 mL to 0.188 g/100 mL. The ethanol positivity rate for postmortem casework remained relatively the same at approximately 20% during the time frame, while there was a decrease in ethanol positivity rate for DWI casework during the pandemic in April - June (Q2) 2020. However, the positivity rate returned to pre-pandemic levels by the end of 2020. Despite the self-reported surveys of increased alcohol consumption during the pandemic, a corresponding increase in average ethanol concentrations was not observed in Dallas County and the surrounding area., Competing Interests: none., (© 2023 Published by Elsevier B.V.)

The opioid epidemic resulted in an increase in identifications of fentanyl, fentanyl analogs, and heroin, among other opioids, in the toxicology and seized drug analysis fields over the past several years. The Dallas County Southwestern Institute of Forensic Sciences, comprised of the Toxicology Laboratory and Drug Analysis Laboratory, observed a rise in fentanyl-positive analyses in both laboratories. Fentanyl positivity increased from 1.1% to 3.4% in the Toxicology Laboratory over the five years of this study, whereas 6-monoacetylmorphine (6-MAM) positivity remained relatively stable at approximately 2%. Similarly, in the Drug Analysis Laboratory, the fentanyl positivity rate changed from 0.03% to 0.60%, whereas heroin identification remained stable at approximately 8-9%. Based on data obtained from toxicology submissions, a typical fentanyl or heroin user was a white male in their late 30s. The average concentration of fentanyl in postmortem cases, antemortem cases, and 6-MAM in postmortem specimens was 9.7 ng/ml, 5.5 ng/ml, and 68.4 ng/ml, respectively. Poly-drug use was evident, with benzodiazepines being the most commonly co-administered drug with fentanyl (49.4%). Multiple drugs were identified via analysis of clandestine tablets submitted to the Drug Analysis Laboratory, including fentanyl and/or heroin in combination with compounds such as cocaine, etizolam, and acetaminophen. The most frequently identified clandestine tablet was "M367," typically manufactured to contain acetaminophen and hydrocodone, but was found to contain fentanyl or heroin. The results from this study aid in the understanding of the current opioid trends in Dallas County and provides an opportunity for comparison to other populations and geographical regions., (© 2022 American Academy of Forensic Sciences.)

Drug-facilitated sexual assault is a form of sexual violence against an individual incapacitated by alcohol and/or drugs consumed voluntarily or covertly administered. The purpose of this study was to evaluate toxicological results and the associated demographics of sexual assault-related cases submitted to Houston Forensic Science Center from 2014 to 2020. In total, 1240 samples (1230 cases) were tested during the six-year period that consisted of blood, urine, or both specimens. Blood was analyzed for ethanol by dual-column headspace gas chromatography with flame ionization detection. Drug screen analysis was performed preferably on urine specimens using enzyme-linked immunosorbent assay. Positive screening results were confirmed upon request only due to laboratory policy. A total of 22% (n = 176) of requested samples were confirmed positive. Ethanol was the most prevalent substance detected, present in 17% of the samples (n = 212), followed by 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) (n = 118). The combination of ethanol and THC-COOH was the most frequent one found (n = 17) in cases positive for two or more drugs (n = 101). Demographic data showed the majority of DFSA victims were white (25%) females (72%) with an average age of 27 years old (n = 348). Almost 90% of cases where the presence of drugs was confirmed resulted in no charges being made, either due to lack of suspect information or unknown reasons by the laboratory., (© 2022 American Academy of Forensic Sciences.)

Drug-impaired driving is a growing public safety issue. Addressing impairment due to drugs other than ethanol can be challenging for forensic toxicologists as many factors need to be considered including the type of drug(s), drug-drug interaction, the dose(s) and the individual's physiological condition and drug use history. Interpretation of blood drug test results is additionally difficult as drug concentrations in impaired driving cases may overlap levels typically viewed as toxic. This study compares blood concentrations of drugs in impaired driving cases to those in postmortem cases in Houston, TX, from 2014 to 2020. Blood drug concentrations from driving while intoxicated (DWI) or driving under the influence of drugs (DUID) cases submitted to Houston Forensic Science Center (HFSC) and Harris County Institute of Forensic Sciences (HCIFS) were compared to postmortem blood test results from HCIFS. Eight DWI/DUID cases had drugs that exceeded impaired driving concentrations reported in the literature. These drugs included fentanyl (220 ng/mL), oxycodone (680 ng/mL), hydrocodone (310 and 490 ng/mL), clonazepam (330 ng/mL), methamphetamine (3,500 and 7,100 ng/mL) and tetrahydrocannabinol (THC) (160 ng/mL). For oxycodone and hydrocodone, the presented DWI/DUID cases exceeded 91% and 96% of postmortem concentrations, respectively. The 7,100 ng/mL methamphetamine DWI/DUID result was greater than 98% of postmortem cases. The presented DWI/DUID concentrations were higher than all but one postmortem case for clonazepam and higher than all postmortem cases for THC. This study demonstrates that extremely high drug concentrations in DWI/DUID casework blur the line between therapeutic/recreational and toxic concentrations., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na + transport in isolated mouse TAL tubules was studied using the Na + -sensitive dye, SBFI-AM. Switching from 0 Na + solution to control Ringer's solution (CR) rapidly increased TAL cell [Na + ] i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na + -K + -ATPase inhibitor. This [Na + ] i -elevating effect of CP was time-dependent, required the presence of Na + in the bathing solution, and was insensitive to Na + -K + -2Cl - cotransporter inhibition. Addition of PF to CR elevated [Na + ] i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na + ] i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na + ] i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na + transport in TALs by inhibiting Na + exit via Na + -K + -ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na + -K + -ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The kidneys play an important role in the long-term regulation of blood pressure by control of salt and water balance in the body through various systems including the endocannabinoid system. The endocannabinoid system consists of the two major cannabinoid receptor agonists, anandamide (AEA) and 2-arachidonylglycerol (2-AG), their hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid receptors, CB 1 and CB 2 . AEA can be converted into 12- and 15(S)-hydroperoxyeicosatetraenoic acid ethanolamides by 12-LOX and 15-LOX, respectively and can form epoxyeicosatrienoic acid- (EET-EAs) (5,6-, 8,9-, 11,12-, 14,15-) and hydroxyeicosatetraenoic acid- (HETE) ethanolamides. Furthermore, the EET-EAs produce a secondary metabolism by microsomal epoxide hydrolase to form the corresponding dihydroxyeicosatetraenoic acid-EAs (DiHETE-EA). Reference material was not available for DiHETE-EA. These metabolites were synthesized by incubation of the corresponding EET-EAs with mouse liver cytosol containing epoxide hydrolases. Presented is a solid phase extraction and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the extraction and quantitation of AEA, 2-AG, their metabolites, oleoylethanolamide (OEA), and palmitoylethanolamide (PEA), and the in vivo formation of the DiHETE-EAs in kidney after a single intravenous bolus administration of 20 mg/kg of anandamide in C57BL/6 J and FAAH KO mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The NLRP3 inflammasome is activated in the cytoplasm of cells and its products such as IL-1β are exported through a non-classical ER-Golgi pathway. Several mechanistically distinct models including exocytosis of secretory lysosomes, microvesicles (MVs) and extracellular vehicles (EVs) have been proposed for their release. In this study, we hypothesized that the NLRP3 inflammasome product, IL-1β in response to exogenously administrated and endogenously produced d-ribose stimulation is released via extracellular vesicles including EVs via a sphingolipid-mediated molecular mechanisms controlling lysosome and multivesicular body (MVB) interaction. First, we demonstrated that both endogenous and exogenous d-ribose induced NLRP3 inflammasome activation to produce IL-1β, which was released via EVs in podocytes. Then, we found that colocalization of marker MVB marker VPS16 with IL-1β within podocytes increased upon d-ribose stimulation, which was accompanied by decreased colocalization of lysosome marker Lamp-1 and VPS16, suggesting decrease in MVB inclusion of IL-1β due to reduced lysosome and MVB interaction. All these changes were mimicked and accelerated by lysosome v-ATPase inhibitor, bafilomycin. Moreover, ceramide in podocytes was found elevated upon d-ribose stimulation, and prior treatments of podocyte with acid sphingomyelinase (Asm) inhibitor, amitriptyline, acid ceramidase (AC) inducer, genistein, or AC CRISPR/cas9 activation plasmids were found to decrease d-ribose-induced ceramide accumulation, EVs release and IL-1β secretion due to reduced interactions of lysosome with MVBs. These results suggest that inflammasome-derived products such as IL-1β during d-ribose stimulation are released via EVs, in which lysosomal sphingolipid-mediated regulation of lysosome function plays an important role., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not yet understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured, and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase, PF-3845 ( N -3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide). Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. High-performance liquid chromatography (HPLC) analyses of lipid extracts of MMIC culture medium revealed a 205-nm absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 area units under the peak/10 min) stimulated diuresis and natriuresis, whereas these parameters returned toward baseline at higher doses but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, the glomerular filtration rate, and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urine parameters. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid that acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The kidneys contribute to the control of body fluid and electrolytes and the long-term regulation of blood pressure through various systems, including the endocannabinoid system. Previously, we showed that inhibition of the two major endocannabinoid-hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, in the renal medulla increased the rate of urine excretion (UV) and salt excretion without affecting mean arterial pressure (MAP). The present study evaluated the effects of a selective FAAH inhibitor, N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidine carboxamide (PF-3845) on MAP and renal functions. Infusion of PF-3845 into the renal medulla of C57BL/6J mice reduced MAP during the posttreatment phases and increased UV at 15 and 30 nmol/min per gram kidney weight (g kwt), relative to the pretreatment control phase. Intravenous PF-3845 administration reduced MAP at the 7.5, 15, and 30 doses and increased UV at the 15 and 30 nmol⋅min -1 ⋅g -1 kwt doses. PF-3845 treatment elevated sodium and potassium urinary excretion and medullary blood flow. Homozygous FAAH knockout mice were refractory to intramedullary PF-3845-induced changes in MAP, but UV was increased. Both MAP and UV responses to intramedullary PF-3845 in C57BL/6J mice were diminished by pretreatment with the cannabinoid type 1 receptor-selective antagonist, rimonabant (3 mg/kg, ip) but not the cyclooxygenase 2-selective inhibitor, celecoxib (15 mg/kg, iv). Liquid chromatography-tandem mass spectrometry analyses showed increased anandamide in kidney tissue and 2-arachidonoyl glycerol in plasma after intramedullary PF-3845. These data suggest that inhibition of FAAH in the renal medulla leads to both a diuretic and blood pressure-lowering response mediated by elevated anandamide in kidney tissue or 2-arachidonoyl glycerol in plasma.

Cocaine continues to be one of the most widespread abused illicit drugs in the USA. Rapid methods are needed for the identification and quantitation of cocaine and its metabolites, benzoylecgonine (BE), ecgonine methyl ester (EME) and cocaethylene (CE), in biological specimens by clinical and forensic toxicology laboratories. Presented is a differential ion mobility spectrometry-tandem mass spectrometry (DMS-MS-MS) method for the analysis of cocaine and its major metabolites in human serum that requires minimal sample preparation and no column chromatography. A Shimadzu Nexera X2 ultra-high performance liquid chromatography system was used to infuse the samples into the DMS cell at a rate of 30 μL/min. Separation of cocaine and its metabolites were performed in a SelexION DMS component from Sciex coupled to a QTRAP 6500 with an IonDrive Turbo V source for TurbolonSpray® using acetonitrile as a chemical modifier. Analysis consisted of ramping the CoV from -35 V to -6 V while monitoring the multiple reaction monitoring (MRM) transitions of each analyte. The assay was evaluated for linearity, bias, precision, carryover, interferences and stability. Calibration curves ranged from 10 to 1,000 ng/mL with linear regression correlation coefficients (r2) of 0.9912 or greater for each analyte. The limit of quantitation was set at 10 ng/mL. Intra-day precision (%CV) ranged from 0% to 15% for cocaine, 1% to 19% for BE, 1% to 17% for EME and 0% to 18% for CE. Inter-day precision ranged from 9% to 14% for cocaine, 2% to 17% for BE, 5% to 11% for EME and 5% to 15% for CE. No carryover or interferences were detected. Bland-Altman analysis of previously analyzed specimens by UPLC-MS-MS showed variability of 30% or less. The method demonstrates the applicability of DMS-MS-MS for high throughout analysis of drugs and their metabolites in clinical and forensic toxicology laboratories.

The gasotransmitters are a family of gaseous signaling molecules which are produced endogenously and act at specific receptors to play imperative roles in physiologic and pathophysiologic processes. As a well-known gasotransmitter along with hydrogen sulfide and carbon monoxide, nitric oxide (NO) has earned repute as a potent vasodilator also known as endothelium-derived vasorelaxant factor (EDRF). NO has been studied in greater detail, from its synthesis and mechanism of action to its physiologic, pathologic, and pharmacologic roles in different disease states. Different animal models have been applied to investigate the beneficial effects of NO as an antihypertensive, renoprotective, and antihypertrophic agent. NO and its interaction with different systems like the renin⁻angiotensin system, sympathetic nervous system, and other gaseous transmitters like hydrogen sulfide are also well studied. However, links that appear to exist between the endocannabinoid (EC) and NO systems remain to be fully explored. Experimental approaches using modulators of its synthesis including substrate, donors, and inhibitors of the synthesis of NO will be useful for establishing the relationship between the NO and EC systems in the cardiovascular and renal systems. Being a potent vasodilator, NO may be unique among therapeutic options for management of hypertension and resulting renal disease and left ventricular hypertrophy. Inclusion of NO modulators in clinical practice may be useful not only as curatives for particular diseases but also for arresting disease prognoses through its interactions with other systems.

The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30, and 60 nmol·min -1 ·kg -1 ) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine whether endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide nor IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg·kg -1 ·30 min -1 iv) nor intramedullary (15 nmol·min -1 ·kg -1 ·30 min -1 ) IDFP pretreatment before intramedullary anandamide (15-30 nmol·min -1 ·kg -1 ) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary PGE 2 concentrations by HPLC-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE 2 These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney., (Copyright © 2017 the American Physiological Society.)

Tianeptine (7-[([3-chloro-6,11]-dihydro-6-methyldibenzo[c,f][1,2]thiazepin-11-yl) amino] heptanoic acid S, S dioxide) is a tricyclic compound prescribed as an antidepressant in European countries, but is not currently approved for use in the United States. There are few published case reports of tianeptine intoxication. Presented is the first case of acute toxicity associated with the intravenous use of tianeptine. A 36-year-old male intentionally injected tianeptine powder intravenously to "help him see into the future". He became unresponsive and a bystander called emergency medical services. Upon arrival to the Emergency Department, excessive constriction of the pupils, sedation, and a respiratory rate of 6 respirations per minute (rpm) were noted. Blood and urine were collected ~2 h post admission. The patient's serum ethanol concentration was 133 mg/dL. His toxicity was successfully reversed with two doses of naloxone 0.4 mg IV. He was started on a naloxone infusion at 0.2 mg/h and discharged 13 h after admittance awake, alert and oriented. The patient's urine sample screened negative for common drugs of abuse and total tricyclic antidepressants. A high performance liquid chromatography tandem mass spectrometry method was developed and validated to quantify tianeptine in urine. The calibration range was 1-100 ng/mL with linear regression correlation (r2) of 0.9996 or greater. The limit of quantitation was administratively set at 1 ng/mL. The bias of the assay was determined to be within ±20% of the target value for each quality control specimen. The intra-day and inter-day precision did not exceed 15% coefficient of variation for each quality control specimen. Matrix effects, absolute recovery, carryover and specificity were also evaluated. The patient's tianeptine urine concentration was determined to be 2 ng/mL., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)