Microarray characterization of gene expression changes in blood during acute ethanol exposure.

BACKGROUND: As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. METHODS: Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGene™ blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. RESULTS: Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR.The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. CONCLUSIONS: The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance.

Oral fluid for the detection of drugs of abuse using immunoassay and LC-MS/MS.

The utility of oral fluid as a sample matrix for the analysis of drugs has been increasing in popularity over the last few years. This is largely because of collection advantages over other matrices, but also due to the rapid improvements in analytical assays including highly sensitive liquid reagent format enzyme immunoassays and LC-MS/MS. This review will highlight improvements in assay formats, sensitivity, laboratory equipment and sample processing using low sample volumes to expand drug test profiles.

Prevalence of synthetic cannabinoids in U.S. athletes: initial findings.

A number of synthetic cannabinoids such as JWH-018 and JWH-073 have been incorporated into "spice" products. Despite having labels warning against human consumption, the products are smoked for their cannabinoid-like effects and the extent of their use by athletes has not been adequately described. Urine samples collected from 5,956 athletes were analyzed by high-performance liquid chromatography-tandem mass spectrometry for the presence of JWH-018, JWH-073, and their metabolites. Metabolites of JWH-018 and/or JWH-073 were detected in 4.5% of the samples. Metabolites of JWH-018 and JWH-073, only JWH-018, and only JWH-073 were detected in 50%, 49%, and approximately 1% of positive samples, respectively. In total, JWH-018 metabolites were detected in 99% (50% + 49%) and JWH-073 metabolites were detected in approximately 50% (49% + 1%) of the positive samples. Parent JWH-018, JWH-018-2-OH-indole, and JWH-018-4-OH-indole were not detected in any of the samples. All samples in which JWH-073 metabolites were detected contained JWH-073-N-butanoic acid. Parent JWH-073 and its N-(4-OH-butyl), 4-OH-indole, 5-OH-indole, and 7-OH-indole metabolites were not detected. Given the number of synthetic cannabinoids that have been synthesized, their limited regulation, and the prevalence of JWH-018 and JWH-073 metabolites detected in the athletes, these compounds should remain a priority for anti-doping programs.

The determination of cannabinoids using liquid chromatography with mass spectrometric detection.

Because of their prevalence in drugged driving and other medicolegal investigations, cannabinoids are routinely analyzed by forensic laboratories. Until relatively recently, these analyses were performed by gas chromatography coupled to mass spectrometry (GC-MS). However, the need for derivatization and extensive sample preparation made GC-MS approaches tedious and time consuming. As a consequence, many laboratories have explored alternative analysis techniques. The advent of more affordable liquid chromatography-mass spectrometry (LC-MS) instruments and the utility of atmospheric pressure ionization sources have made LC-MS a promising alternative to GC-MS for the detection and quantitation of cannabinoids in forensic applications.

Oral fluid drug testing of chronic pain patients. II. Comparison of paired oral fluid and urine specimens.

A clinical study was conducted to compare the use of oral fluid to urine for compliance monitoring of pain patients. Patients (n = 133) undergoing treatment for chronic pain at four clinics participated in the study and provided paired oral fluid and urine specimens. Oral fluid specimens were collected with Quantisal(TM) saliva collection devices immediately following urine collection. Oral fluid specimens were analyzed for 42 drugs and/or metabolites by validated liquid chromatography-tandem mass spectrometry procedures. Accompanying urine specimens were initially screened by immunoassay and non-negative results were confirmed. Of the 1544 paired tests, 329 (21.3%) drug analytes were positive, and 984 (63.7%) were negative for both specimens resulting in an overall agreement of 85%. There were 83 (5.4%) analyte results that were positive in oral fluid and negative in urine, and 148 (9.6%) were negative in oral fluid and positive in urine for an overall disagreement of 15%. Cohen's Kappa value was 0.64, indicating "substantial" agreement. The primary exceptions to agreement were the lower detection rates for hydromorphone, oxymorphone, and benzodiazepines in oral fluid compared to urine. The authors conclude that, overall, oral fluid tests produced comparable results to urine tests with some minor differences in detection rates for different drug classes.

Screening indicators of dehydroepiandosterone, androstenedione, and dihydrotestosterone use: a literature review.

Because of their perceived and reported effects on self-image, muscle development, performance, and similar factors, anabolic-androgenic steroids (AAS) and their precursors are among the most abused substances by professional, amateur, and recreational athletes. However, AAS abuse is not limited to athletes, but is also prevalent in the workplace, especially those professions in which image, strength, and endurance are coveted attributes. The detection of many steroids in biological specimens is analogous to the detection of an abused drug such as cocaine. Identification of the parent drug or its characteristic metabolite(s) in a donor's sample with a drug screening technique and confirmation of the drug/metabolite with a suitable alternative technology provides evidence of use. These analyses and subsequent interpretive scenarios become far more complex when the ingested AAS is an endogenous compound such as dehydroepiandrosterone (DHEA), androstenedione (Adione), or dihydrotestosterone (DHT). These compounds and their metabolites are present in specimens such as urine as a course of our natural endocrine function. Therefore, it becomes much more challenging for the laboratory to establish testing and interpretative paradigms that can distinguish "normal" urinary profiles of these steroids and their metabolites from profiles indicative of exogenous use. Distinguishing "normal" from "abnormal" urine profiles is particularly challenging during screening when literally tens of steroids and their metabolites may be tested simultaneously in a single chromatographic analysis. The purpose of this paper is to review the relevant literature about DHEA, Adione, and DHT administration, detection, and interpretation specifically as it relates to changes in the urinary AAS profile that may be identified during the routine laboratory screening of donor urine specimens.

Rapid urine drug screens: diphenhydramine and methadone cross-reactivity.

BACKGROUND: Rapid urine screens to detect drugs of abuse are often used in pediatric emergency departments (PEDs). A positive result may lead to further clinical testing, social evaluation, and increased stress/inconvenience. A PED patient with suspected diphenhydramine (DPH) ingestion had a positive methadone result on the rapid urine drug screen, One Step Multi-Drug, Multi-Line Screen Test Device (ACON Laboratories, San Diego, Calif). There was no history of methadone exposure so the patient was admitted while confirmatory testing was performed. Gas chromatography/mass spectroscopy testing of the urine failed to confirm the presence of methadone. We present this unreported false-positive methadone result and evaluation of the kit for cross-reactivity of DPH and methadone. METHODS: The same One Step urine drug screen was tested at an independent laboratory for cross-reactivity between methadone and DPH including the DPH metabolites. Drug-free urine was fortified with DPH, nordiphenhydramine, or dinordiphenhydramine at 0, 10, 25, 50, and 100 µg/mL for each analyte. One hundred microliters of the solutions were added to each of the 4 wells on test cassettes. Urine was allowed to migrate according to manufacturer instructions. Each cassette was interpreted by 2 analysts to ensure consistent interpretation and accurate data recording. RESULTS: In vitro laboratory testing results showed cross-reactivity between methadone and DPH but not for nordiphenhydramine or dinordiphenhydramine. CONCLUSIONS: Rapid urine drug screens using immunoassays based on the principle of competitive binding may show false-positive methadone results for patients who have ingested DPH. Product information for urine drug screens may not include all cross-reacting agents and should be used with caution when interpreting drug screen results in PED patients.

Laboratory evaluation and field application of roadside oral fluid collectors and drug testing devices.

This study was a part of a collaborative U.S./E.U. international research effort (Roadside Testing Assessment, ROSITA II) to assess illegal drug use among motor vehicle operators suspected of driving while under the influence of drugs and to evaluate the effectiveness of point-of-collection oral fluid drug detection technologies. A goal of the study was to assess commercial oral fluid drug testing devices for potential use in law enforcement. Ten devices were evaluated in the laboratory for their ability to meet manufacturers' claimed (and proposed) cutoff concentrations for the detection of amphetamine(s), cocaine/metabolite, opiates, cannabinoids, and benzodiazepines (2 devices). The field study portion of the research was conducted in major cities in the United States and Western Europe by teams of scientists working in collaboration with the local police. In Salt Lake City, Utah, the Drugwipe, Securetec, Ottobrunn, Germany (Securetec) oral fluids drug testing device was also evaluated in the field by testing suspected drug-impaired drivers. During the initial phase of the field study, 40 subjects were recruited. Drugwipe results were compared with laboratory-based immunoassay and mass spectrometry results and demonstrated that calculated sensitivities were between 75% and 100% depending on drug class. Specificities varied from 36% for cannabinoids to over 95% for opiates. During the second phase of the field study, 267 subjects were recruited. The Drugwipe sensitivities were 36.4%, 35.9%, 42.9%, and 7.7%, respectively, for amphetamine(s), cocaine, opiates, and cannabinoids. The Drugwipe specificities were 99.2%, 97.4%, 99.6%, and 99.6%, respectively, for amphetamine(s), cocaine, opiates, and cannabinoids. Drugwipe failed to meet the study criteria for acceptable device performance, required performance sensitivities, and specificities 90% or greater.

Detection of bumetanide in an over-the-counter dietary supplement.

Bumetanide is a loop diuretic used clinically to treat heart failure, acute renal failure, high blood pressure, and edema. However, diuretics may also be used by athletes as masking agents and to decrease weight. Taken as masking agents, diuretics increase urine production and decrease urinary concentrations of banned performance-enhancing agents, such as anabolic steroids. StarCaps is an over-the-counter dietary supplement marketed as a diet aid. The manufacturer claims that the product contains only natural cleansing agents and emphasizes that it is free from traditional appetite suppressants such as sympathomimetic amines. However, no such disclaimer is made concerning diuretic agents. A single StarCaps capsule was administered to two male and two female volunteers, and their urine specimens were collected at discrete intervals (2, 4, 8, and 12 h) post administration. The specimens were analyzed by a high-performance liquid chromatography-mass spectrometry quadrupole (HPLC-MS) method, and bumetanide was detected in all specimens (4.6 to 351.3 ng/mL). Adjusting the bumetanide concentrations for creatinine content did little to normalize the excretion profiles. Bumetanide was also detected in the StarCaps capsules at concentrations approaching therapeutic doses. HPLC-quadrupole-time-of-flight mass spectrometry was used to confirm the presence of bumetanide in the urine samples and StarCaps capsules. The results showed that unregulated dietary supplements may put consumers at risk for unwitting consumption of prescription medications, and that it is possible for athletes to inadvertently test positive for bumetanide and face disciplinary actions.

Analysis of a challenging subset of World Anti-Doping Agency-banned steroids and antiestrogens by LC-MS-MS.

A qualitative liquid chromatography-tandem mass spectrometry method for the analysis of 22 sporting federation-banned anabolic agents (or their metabolite markers) and anti-estrogens in urine that are refractory to analysis by gas chromatography-mass spectrometry is presented. In addition, a quantitative method built around World Anti-Doping Agency (WADA) guidelines for the confirmatory analysis of 19-norandrosterone, the primary metabolite of nandrolone with a WADA-specified minimum required performance limit of 1 ng/mL, is included. Hydrolysis of glucuronide conjugates, liquid-liquid extraction, no clean-up derivatization with Girard's Reagent P, and analysis by quadrupole-time-of-flight mass spectrometry provide sensitivity and selectivity well beyond that required by the WADA.

A validated method for the detection of Delta 9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta 9-tetrahydrocannabinol in oral fluid samples by liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

A quantitative liquid chromatography-electrospray ionization-quadrupole-time-of-flight (TOF) mass spectrometry (MS) method for simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in oral fluid samples was developed and fully validated. The analytes were isolated from the oral fluid by classic liquid-liquid extraction. The two compounds were separated in 9.5 min with a total analysis time of 19 min. The linear dynamic range was established from 0.1 to 100 ng/mL for THC and 0.5-100 ng/mL for the THC-COOH. The method had a good intra- and interassay accuracy and precision at each measured concentration. No matrix effects were observed for the analytes or the labeled internal standards. Accurate mass measurement was achieved to +/- 5 ppm. The sensitivity of the method results from a combination of unique chromatographic retention time, the use of MS-MS, and the "exact mass" measurement of the target ions by a TOF analysis. The method was successfully applied to two types of oral fluid samples. One type was collected from roadside driving-under-the-influence studies. The second was used to assess the recovery of the THC from an oral fluid collection device under laboratory conditions. Although the use of the TOF MS in the forensic and toxicology fields is not as widespread as the triple-quadrupole technology, it offers some significant analytical advantages that are demonstrated by this method.

Evaluation of ten oral fluid point-of-collection drug-testing devices.

Previously, the laboratory evaluations of six point-of-collection oral fluid (POC-OF) drug testing devices were reported. Four additional devices, Oralstat (American Bio Medica); SmartClip (Envitec); Impact (LifePoint); and OraLine IV s.a.t (Sun Biomedical Laboratories), were recently evaluated for their ability to meet the claimed (and proposed) cutoff concentrations set by the manufacturers for the detection of amphetamine(s), cocaine/metabolite, opiates, and cannabinoids (Oralstat also benzodiazepines). With the exception of the Sun Biomedical device, actual false-positive results were not encountered. Most devices performed well for the detection of opiates and amphetamine(s), but approximately half had amphetamine(s) cutoff concentrations greater than that proposed by the Substance Abuse and Mental Health Services Administration (SAMHSA). Only three devices had cocaine cutoffs less than or equal to 20 ng/mL (SAMHSA), and a number of false-negative results were obtained. The devices still were not capable of detecting Delta(9)-tetrahydrocannabinol at 4 ng/mL (SAMHSA). However, sensitivities improved since the initial studies, and approximately half of the devices met the THC-COOH cutoff proposed by SAMHSA. Results from the current and previous evaluations are presented in the paper and indicate that the sensitivity and performance of commercial OF drug testing devices is improving, but remains problematic for the reliable detection of cannabinoid use.

Recovery of drugs of abuse from the Immunalysis Quantisal oral fluid collection device.

Drug recovery from a new oral fluid collection device was assessed. The evaluation was performed in vitro at three physiologically relevant concentrations for the following substances: amphetamine, methamphetamine, morphine, codeine, cocaine, benzoylecgonine, methadone, oxazepam, and Delta(9)-tetrahydrocannabinol (THC). Drug-free and drug-fortified controls were prepared and their concentration verified by liquid chromatography-tandem mass spectrometry. Aliquots of the controls were then "collected" with the device (n=3) using the manufacturer's recommended procedure. Collected samples were stored for 12 h to simulate shipping before analysis. Fresh, non-"collected" aliquots of each pool (n=3) were concurrently analyzed. The drug recoveries from the Quantisal were expressed as a mean percentage of the concurrently analyzed aliquots that were not subjected to device collection. Recoveries for oxazepam exceeded 97%, for amphetamine and methamphetamine exceeded 93%, and for opioids all exceeded 91%. The recoveries of cocaine were >91% and >82% for its polar metabolite, benzoylecgonine. Especially noteworthy was the recovery of THC from the Quantisal collector (81.3-91.4%). When compared with recoveries reported from other collection devices, the Quantisal was clearly superior.

A comparison of the cell phone driver and the drunk driver.

OBJECTIVE: The objective of this research was to determine the relative impairment associated with conversing on a cellular telephone while driving. BACKGROUND: Epidemiological evidence suggests that the relative risk of being in a traffic accident while using a cell phone is similar to the hazard associated with driving with a blood alcohol level at the legal limit. The purpose of this research was to provide a direct comparison of the driving performance of a cell phone driver and a drunk driver in a controlled laboratory setting. METHOD: We used a high-fidelity driving simulator to compare the performance of cell phone drivers with drivers who were intoxicated from ethanol (i.e., blood alcohol concentration at 0.08% weight/volume). RESULTS: When drivers were conversing on either a handheld or hands-free cell phone, their braking reactions were delayed and they were involved in more traffic accidents than when they were not conversing on a cell phone. By contrast, when drivers were intoxicated from ethanol they exhibited a more aggressive driving style, following closer to the vehicle immediately in front of them and applying more force while braking. CONCLUSION: When driving conditions and time on task were controlled for, the impairments associated with using a cell phone while driving can be as profound as those associated with driving while drunk. APPLICATION: This research may help to provide guidance for regulation addressing driver distraction caused by cell phone conversations.

Assessment of pepper spray product potency in Asian and Caucasian forearm skin using transepidermal water loss, skin temperature and reflectance colorimetry.

Historically, pepper spray product potency has been established using a taste test evaluation. A taste test is subjective and may not be appropriate for assessing pepper potency in skin. The current study evaluated chemically diverse pepper sprays in human forearm skin using three objective, noninvasive parameters: transepidermal water loss, skin surface temperature and erythema, as a means for assessing dermal pharmacology, toxicology and product potency. Five commercial pepper spray products containing various capsaicinoid analogs at various concentrations were evaluated in duplicate on volar forearms of six Caucasians and six Asians using a 10 min exposure. Mean surface skin temperature, transepidermal water loss results were highly variable and therefore did not demonstrate dose responsive behavior to increasing capsaicinoid concentrations. Erythema, as measured by increases in a* (reflected light in the red-to-green color spectrum) of the L*a*b* uniform color scale, was superior among parameters evaluated in discriminating pepper spray potency and correlated well with the relative and total capsaicinoid concentration in the products. Products containing greater than 16 mg ml(-1) capsaicinoid concentration produced greater erythema responses in Caucasians than Asians. Asians responded greater to the synthetic analog, nonivamide, than to mixtures of capsaicinoids, while Caucasians responded equally to both capsaicinoid analogs. Thus, pepper spray product potency in human skin reflects the total capsaicinoid concentration, the specific capsaicin analog(s) present, and the race of the individual exposed. The finding that the reflectance colorimeter a* scale can differentiate these parameters in skin will have a significant impact on evaluating the use and efficacy of pepper spray products in humans.

An evaluation of selected oral fluid point-of-collection drug-testing devices.

Point-of-collection oral fluids drug-testing devices are being marketed for a variety of medico-legal purposes where they may complement existing technologies and be used to detect drugs following recent ingestion. To assess the utility of these devices for use in drugged-driving investigations, we performed a laboratory evaluation of four devices and those results were published previously. In the study reported here, two more devices, Oratect(R) (Branan) and Uplink(R) (OraSure), were evaluated for their ability to detect amphetamines, cocaine, opiates, and cannabinoids. An additional device, Drugwipe (Securtec), was evaluated for the detection of cocaine and cannabinoids. Each of the devices was assessed for their ability to meet the manufacturers' claimed cutoff concentrations and to meet cutoffs proposed for federal workplace programs. In general, the Branan and OraSure devices detected amphetamine, methamphetamine, opiates, and cannabinoid metabolite (THC-COOH) well in the concentration ranges approximating those proposed by the Substance Abuse and Mental Health Services Administration (SAMHSA), but all three devices performed poorly in detecting Delta9-tetrahydrocannabinol (THC) at the proposed SAMHSA cutoff. The ability to accurately and reliably detect cocaine was dependent on the individual device, and the Branan and Securetec devices were more effective than OraSure at detecting parent cocaine.

Oral fluid collection: the neglected variable in oral fluid testing.

The potential to use oral fluid as a drug-testing specimen has been the subject of considerable scientific interest. The ease with which specimens can be collected and the potential for oral fluid (OF) drug concentrations to reflect blood-drug concentrations make it a potentially valuable specimen in clinical as well as forensic settings. However, the possible effects of the OF collection process on drug detection and quantification has often been over looked. Several studies have documented that drug-contamination of the oral cavity may skew oral fluid/blood drug ratios and confound interpretation when drugs are smoked, insufflated or ingested orally. OF pH is predicted to have an effect on the concentration of drugs in OF. However, in a controlled clinical study, the effect of pH was less than that of collection technique. Mean codeine OF concentrations in specimens collected a non-stimulating control method were 3.6 times higher than those in OF collected after acidic stimulation. Mean codeine concentrations were 50% lower than control using mechanical stimulation and 77% of control using commercial collection devices. Several factors should be considered if a commercial OF collection device is used. In vitro collection experiments demonstrated that the mean collection volume varied between devices from 0.82 to 1.86 mL. The percentage of the collected volume that could be recovered from the device varied from 18% to 83%. In vitro experiments demonstrated considerable variation in the recovery of amphetamines (16-59%), opiates (33-50%), cocaine and benzoylecgonine (61-97%), carboxy-THC (0-53%) and PCP (9-56%). Less variation in collection volume, volume recovered and drug recovery was observed intra-device. The THC stability was evaluated in a common commercial collection protocol. Samples in the collection buffer were relatively stable for 6 weeks when stored frozen. However, stability was marginal under refrigerated conditions and poor at room temperature. Very little has been published on the efficacy of using IgG concentration, or any other endogenous marker, as a measure of OF specimen validity. Preliminary rinsing experiments with moderate (50 mL and 2 x 50 mL) volumes of water did not reduce the OF IgG concentration below proposed specimen validity criteria. In summary, obvious and more subtle variables in the OF collection may have pronounced effects on OF-drug concentrations. This has rarely been acknowledged in the literature, but should to be considered in OF drug testing, interpretation of OF-drug results and future research studies.

Age-dependent methamphetamine-induced alterations in vesicular monoamine transporter-2 function: implications for neurotoxicity.

Tens of thousands of adolescents and young adults have used illicit methamphetamine. This is of concern since its high-dose administration causes persistent dopaminergic deficits in adult animal models. The effects in adolescents are less studied. In adult rodents, toxic effects of methamphetamine may result partly from aberrant cytosolic dopamine accumulation and subsequent reactive oxygen species formation. The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine into synaptic vesicles for storage and perhaps protection against dopamine-associated oxidative consequences. Accordingly, aberrant VMAT-2 function may contribute to the methamphetamine-induced persistent dopaminergic deficits. Hence, this study examined effects of methamphetamine on VMAT-2 in adolescent (postnatal day 40) and young adult (postnatal day 90) rats. Results revealed that high-dose methamphetamine treatment caused greater acute (within 1 h) decreases in vesicular dopamine uptake in postnatal day 90 versus 40 rats, as determined in a nonmembrane-associated subcellular fraction. Greater basal levels of VMAT-2 at postnatal day 90 versus 40 in this purified fraction seemed to contribute to the larger effect. Basal tissue dopamine content was also greater in postnatal day 90 versus 40 rats. In addition, postnatal day 90 rats were more susceptible to methamphetamine-induced persistent dopaminergic deficits as assessed by measuring VMAT-2 activity and dopamine content 7 days after treatment, even if drug doses were adjusted for age-related pharmacokinetic differences. Together, these data demonstrate dynamic changes in VMAT-2 susceptibility to methamphetamine as a function of development. Implications with regard to methamphetamine-induced dopaminergic deficits, as well as dopamine-associated neurodegenerative disorders such as Parkinson's disease, are discussed.