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Stratified medicine is the tailoring of treatment to the individual characteristics of each patient. This is a challenging task in the context of psoriasis, a complex disease with a variety of phenotypic presentations and a comorbidity burden that extends beyond cutaneous manifestations. In recent years, considerable progress has been made in understanding the immunology of psoriasis, and this has informed the development of increasingly precise and efficacious therapies. However, not all patients respond to biologic therapy, and access is limited to patients with moderate to severe disease. However, subpopulations of patients are emerging with distinct patterns of response to therapy, largely determined by clinical and pharmacogenomic factors. Despite progress to date, the natural history of psoriasis remains poorly understood. It is likely that disease onset, progression, development of comorbidities and response to therapy are due to a combination of genetic, inflammatory and environmental factors. We envision that a greater understanding of the natural history of psoriasis will be a key factor in progressing a stratified medicine approach to patient care, as will earlier intervention in the course of the disease.

Histone chaperone HIRA is thought to play a role in both early development and aging, but little is known about connections between the two processes. Here, we explore this relationship using a lineage-specific knockout mouse model, TyrCre::Hirafl/fl, in which HIRA is deficient in the pigmentary system consisting of embryonic melanoblasts, postnatal melanocytes and melanocyte stem cells (McSCs). Hira knockout leads to reduced melanoblast numbers during embryogenesis, but wild type numbers of melanocytes at birth, normally functioning juvenile and young adult McSCs, and only a very mildly hypopigmented first hair coat. However, on closer analysis, Hira knockout melanocytic cells of newborn mice exhibit molecular markers characteristic of cell aging and proliferative deficits. As they age, TyrCre::Hirafl/fl mice display marked defects in McSC maintenance and premature hair graying. Importantly, these defects are only observed when HIRA is inactivated during embryogenesis, not post-natally. This genetic model illustrates how normal embryonic development lays the foundation for maintenance of adult tissue specific stem cells and so suppression of degenerative phenotypes of aging.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML., MDM2 and BET inhibitors have shown efficacy in AML treatment. Here, the authors show that the two compounds can synergize through both p53 protein stabilization and inhibition of BRD4-mediated repression of p53 target genes.

Summary Approximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer., Graphical Abstract, Highlights • DNMT3B antagonizes BRAFV600E-induced senescence-like state • DNMT3B accelerates BRAFV600E-induced intestinal cancer • Other studies do not support a role for DNMT3B in CIMP and cooperation with BRAFV600E • On balance, BRAFV600E and DNMT3B are unlikely to cooperate in human intestinal cancer, Biological Sciences; Molecular Biology; Cancer

Background and objectives Patients on in-center dialysis spend significant amounts of time in the dialysis unit; additionally, managing ESRD affects many aspects of life outside the dialysis unit. To improve the care provided to patients requiring hemodialysis, their experiences and beliefs regarding treatment must be understood. This systematic review aimed to synthesize the experiences of patients receiving in-center hemodialysis. Design, setting, participants, & measurements We searched Embase, MEDLINE, CINAHL, PsychINFO, Google Scholar, and reference lists for primary qualitative studies published from 1995 to 2015 that explored the experiences of adult patients receiving treatment with in-center hemodialysis. A thematic synthesis was conducted. Results Seventeen studies involving 576 patients were included in the synthesis. Four analytic themes were developed. The first theme (a new dialysis–dependent self) describes the changes in identity and perceptions of self that could result from dialysis dependence. The second theme (a restricted life) describes the physical and emotional constraints that patients described as a consequence of their dependence. Some patients reported strategies that allowed them to regain a sense of optimism and influence over the future, and these contributed to the third theme (regaining control). The first three themes describe a potential for change through acceptance, adaption, and regaining a sense of control. The final theme (relationships with health professionals) describes the importance of these relationships for in-center patients and their influence on perceptions of power and support. These relationships are seen to influence the other three themes through information sharing, continuity, and personalized support. Conclusions Our synthesis has resulted in a framework that can be used to consider interventions to improve patients’ experiences of in-center hemodialysis care. Focusing on interventions that are incorporated into the established relationships that patients have with their health care professionals may enable patients to progress toward a sense of control and improve satisfaction with care.

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.

Criminologists have always been interested in crimes that involve physical violence. The concept of technology-facilitated coercive control (TFCC) is grounded in the understanding that the social problem of domestic violence (DV) is a pattern of abusive, coercive, and controlling behaviors that occur in the context of profound and persistent familial, cultural, and structural gender inequality. Technology provides a means for perpetrators of DV to enact coercive control that transcends fixed borders and boundaries. Minimization of nonphysical abuse is another barrier to effective responses to TFCC. Digital media have positive as well as negative uses for responding to TFCC. These channels are commonly used for delivering information about services and providing anti-violence education. TFCC is an important aspect of DV and coercive control that merits further attention and requires additional critical research. The monetization of online abuse via traffic-linked marketing is another barrier to effective responses to TFCC.

In 2016, there will be approximately 19,950 new cases of Acute Myeloid Leukaemia (AML) in the United States. After diagnosis, five-year survival is currently ~26%, with available therapeutic approaches. Therefore, there remains a critical requirement for novel therapies for AML. Bromodomain and extra-terminal domain inhibitors (BETi) are emerging as exciting therapeutic agents for haematopoietic malignancies, including AML. The BET protein family, BRD2, BRD3, BRD4 and BRDT, are best known as transcriptional co-activators whose targets include oncogenic loci, such as c-MYC, BCL2 and CDK4/6. Pharmacological inhibition of BET bromodomains targets malignant cells by preventing reading of acetylated lysine residues, thus disrupting chromatin-mediated signal transduction, which reduces transcription at these oncogenic loci. BETi alone have shown promising pre-clinical activity against diverse AML subtypes, and are now in clinical trial in AML and other hematological malignancies. Significantly, however, there is also some evidence that BET family proteins can also act as transcriptional repressors. Whether BET-mediated transcriptional repression is also a therapeutic target in cancer is not known. Although a heterogeneous disease, most human de novo AML (not due to relapse or secondary to myelodysplastic syndrome (MDS) or cancer therapy) harbor wild type TP53 (encoding the p53 tumor suppressor). TCGA reports that only 8% of de novo AML contain TP53 mutations (typically in AML of a complex karyotype). However, p53 is often rendered functionally deficient by over-expression of its negative regulator, MDM2. Accordingly, we hypothesized that dual inhibition of MDM2 and BET would be synergistically lethal to wild type TP53 AML. Confirming this hypothesis, we showed that a combination of MDM2 inhibitors (MDM2i) and BETi is synergistically lethal to human AML cell lines harbouring wild type TP53in vitro, against two mouse models of AML in vivo and against some primary human patient blasts in vitro. Importantly, in the mouse model, the drug combination did not affect normal haematopoiesis, suggesting a manageable level of toxicity. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression both depended on expression of wild type TP53. Dual inactivation by CRISPR/Cas9 of two p53-activated pro-apoptotic genes, PUMA and NOXA, suppressed cell killing by the MDM2i/BETi drug combination. These data indicate that BETi potentiate activation of p53 by MDM2i to kill AML blasts. Regarding the mechanism of synergy, BETi did not enhance binding of p53 to its pro-apoptotic target genes and did not stabilize mRNAs of p53 target genes. Interestingly, however, we observed that in growing human AML cells, a target of BETi, BRD4, was constitutively bound to p53 target genes in control cells and MDM2i-treated cells, but displaced in BETi-treated cells. Accordingly, we hypothesized that BRD4 represses p53 target genes, preventing their activation by MDM2i alone but leading to synergistic activation and cell killing by combined MDM2i and BETi. Consistent with this hypothesis, knock down of BRD4 activated p53 target genes in the presence of MDM2i and ectopic expression of BRD4 repressed p53 target genes. Taken together, these data show that BETi potentiate activation of p53 by MDM2i by relieving BRD4-mediated repression of p53 target genes. This results in potent and specific toxicity towards AML cells harbouring wild type TP53. Disclosures No relevant conflicts of interest to declare.

This study examines key edlements associated with Western firms entering Central and Eastern Europe (CEE): a region classified as transitional. We highlight the role of networking in the foreign market-entry (FME) process in terms of entry strategies. Following a qualitative analysis of six Western (Australian) firms entering two main (Poland and Czech Republic) transitional markets, three key themes were identified: networking strongly underpins initial market-entry; is fundamental in refining target-market decisions; and is integral to strategy-building for market participation in the post-entry phase. Based on our findings we describe a distinct market-situation for emerging markets. We then propose a typology, identifying a fifth “type” of market-situation in emerging markets, the “Transitional International,” an extension of Johnson and Mattsson's (1988) typology. Our ‘Transitional International’ firm is characterised by significant pre-entry and development of post-entry business networks w...

The purpose of this study was to determine the performance characteristics of the Cozart 1 Amphetamine Microplate EIA for detecting amphetamine in oral fluid. Oral fluid samples were collected using the Cozart 1 RapiScan Collection System from 135 volunteer donors from drug treatment clinics. A further 35 oralfluid samples were collected from volunteer donors whowere not drug users. The samples were analyzed in the laboratory using the Cozart 1 Amphetamine Microplate EIA and confirmed using gas chromatography‐mass spectrometry (GC‐MS). The samples were stored frozen until analysis by GC‐MS. The intra-assay precision for the Cozart 1 Amphetamine Microplate EIA for amphetamine in oral fluid over forty assays was 2.74‐7.1% CV (withinassay) and 3.4‐7.0%CV (withinday).Atotalof78sampleswerepositiveforvariousamphetamines and relateddesigner drugs. The Cozart 1 Amphetamine Microplate EIA, using a cutoff of 45 ng/ml amphetamine equivalents in neat oral fluid, had a sensitivity of 91.7 � 3.3% and a specificity of 95.9 � 1.9% versus GC‐MS using a cutoff of 30 ng/ml. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result. # 2005 Elsevier Ireland Ltd. All rights reserved.

PurposeTo provide an Asia‐Pacific viewpoint of the key constraints associated with large geographic distances for smaller westerns firms entering central and eastern Europe (CEE), described as a turbulent transitional environment.Design/methodology/approachAn exploratory study was used within a qualitative methodology, using eight case studies across multiple industries. Semi‐structured interviews were the main method of data collection conducted in 2003/2004. Open, axial and selective coding was used for the analysis to identifying issues.FindingsKey internal constraints for smaller western firms (mindset of western management and middle management in CEE; and lack of management in CEE with decision‐making authority) related to managements' inability to recognize geographic and psychic distance as major external constraints. Largely overcome by enhancing communication between various functional groups; adapting organizational structure; maintaining frequent communicational; developing partnerships in international joint ventures; finding reliable distributors and commitment from re‐sellers and working with government. While no single international business theory adequately explains this process, there is overwhelming support for the network perspective and international entrepreneurship.Research limitations/implicationsThe study is limited by small sample size. The explanatory phase is proposed with further western companies, such as the UK, operating in CEE to identify geographic distance, and additional CEE markets to verify dimensions in this environment.Practical implicationsThe paper provides a checklist of strategies for overcoming constraints facing managers of smaller firms, entering emerging markets with geographic distance.Originality/valuePrevious studies, using a European or Nordic viewpoint, fail to identify the constraints associated with large geographic distances. This paper provides practical assistance to managers starting out in CEE from the Asia‐Pacific.

The purpose of this study was to determine the performance characteristics of the Cozart® microplate EIA for detecting opiates in oral fluid from patients in a drug misuse treatment program. Oral fluid samples were collected using the Cozart® RapiScan Collection System from 216 donors who were receiving treatment for their addiction and were monitored for drug abuse. A further 40 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory by using the Cozart® microplate EIA for opiates and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen until analysis by GC–MS. The intra-assay precision for the Cozart® microplate oral fluid EIA for opiates over 40 assays was 0.43% to 9.13% CV (within assay) and 2.9% to 9.1% CV (within day). A total of 109 samples were positive for various opiates. The Cozart® microplate EIA for opiates in oral fluid, using a cut-off of 30 ng/ml morphine equivalents in neat oral fluid, had a sensitivity of 99.1 ± 2.1% and a specificity of 94.4 ± 2.2% versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

The purpose of this study was to determine the performance characteristics of the Cozart Opiates Microplate ELISA assay for the detection of opiates in hair specimens. One hundred and six hair specimens were collected from volunteers and from drug-related deaths. The hair samples were extracted by sonication followed by overnight extraction in methanol at 60 degrees C. The methanol extract was dried, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture and 0.1M HCl were added to 20 mg of specimen or spiked blank hair and sonicated for 1 h. The opiates were extracted by solid-phase and derivatized with BSTFA + 1% TMS for GC-MS analysis. Fifty-one hair specimens were confirmed positive by GC-MS. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results as the reference method. The optimum cutoff for the Cozart Opiate Microplate ELISA was determined to be between 200 and 300 pg morphine equivalents/mg hair using a 20-mg hair sample. The Cozart Opiates Microplate EIA for opiates in hair using a cutoff of 200 pg/mg hair with a 20-mg hair sample had a sensitivity of 98% +/- 2% and a specificity of 92.7% +/- 3.5% versus GC-MS.

Background: Oral fluid is currently being evaluated as an alternative matrix for monitoring illicit drugs in federally mandated workplace drug testing, for addiction treatment programs, and for driving under the influence testing. The sensitivity, specificity, and efficiency of the Cozart® Microplate EIA Cocaine Oral Fluid Kit (COC ELISA) were determined by comparison with gas chromatography–mass spectrometry (GC/MS) results at screening and confirmation cutoffs proposed in the US and UK. Method: Oral fluid was collected by expectoration after citric acid candy stimulation or with Salivette® neutral cotton swabs or Salivette citric acid-treated cotton swabs before and after cocaine (COC) administration. Specimens (n = 1468) were analyzed with the COC ELISA for screening and with solid-phase extraction followed by GC/MS for confirmation. Three screening cutoffs (10, 20, and 30 μg/L) and four GC/MS cutoffs (2.5, 8, 10, and 15 μg/L COC, benzoylecgonine, and/or ecgonine methyl ester) were evaluated. GC/MS limit of quantification was 2.5 μg/L for all analytes. Results: COC ELISA interassay imprecision (CV; n = 19) was 16% at 16.7 μg/L and 12% at 81.8 μg/L. With the 2.5, 8, 10, and 15 μg/L GC/MS cutoffs, 59.0%, 54.7%, 52.7%, and 48.7% of the oral fluid specimens were positive, respectively. Sensitivity, specificity, and efficiency were 92.2%, 84.7%, and 88.8%, respectively, for the suggested Substance Abuse and Mental Health Services Administration (SAMHSA) cutoffs and 90.2%, 89.2%, and 89.7% for cutoffs currently used in the UK. Conclusions: COC ELISA had suitable sensitivity, specificity, and efficiency for identifying COC exposure at both the proposed SAMHSA and UK cutoffs. Sensitivity, specificity, and efficiency were >84% for both cutoffs, but 92 additional true-positive samples were identified with the SAMHSA cutoffs.

Heartwatch, a structured risk factor modification program for secondary prevention of cardiovascular (CV) disease (CVD) in primary care, is associated with improvements in CV risk factors in participating patients. However, it is not known whether Heartwatch translates into reductions in clinically important CV events.The aim of the study was to determine the association between participation in Heartwatch and future risk of CV events in patients with CVD.The study consisted of a prospective cohort of 1,609 patients with CVD in primary care practices. Of these, 97.5% had data available on Heartwatch participation status, of whom 15.2% were Heartwatch participants. Cox proportional hazards models were used to determine the association between Heartwatch participation and risk of the CV composite (CV death, nonfatal myocardial infarction, heart failure, and nonfatal stroke). All-cause mortality and CV mortality were secondary outcome measures.During follow-up, the CV composite occurred in 208 patients (13.6%). Of Heartwatch participants, 8.4% experienced the CV composite compared with 14.5% of nonparticipants (P = .003). Participation in Heartwatch was associated with a significantly reduced risk of the CV composite (hazard ratio [HR] 0.52, 95% CI, 0.31-0.87), CV mortality (HR 0.31, 95% CI, 0.11-0.89), and all-cause mortality (HR 0.32, 95% CI, 0.15-0.68). Heartwatch participation was also associated with greater reductions in mean systolic blood pressure (P = .047), mean diastolic blood pressure (P.001), and greater use of secondary preventative therapies for CVD, such as lipid-lowering agents (P.001), β-blockers (P.001), and angiotensin-converting enzyme inhibitors (P.001).Heartwatch is associated with a reduced risk of major vascular events and improved risk factor modification, supporting its potential as a nationwide program for secondary prevention of CVD.

The purpose of this study was to determine the performance characteristics of the Cozart Methadone Microplate ELISA assay for the detection of methadone and methadone metabolites in hair specimens. One hundred and ten hair specimens were collected from volunteers (n=46) with a history of drug use and from drug-related deaths (n=64). The hair samples (approximately 20 mg) were extracted by sonication in methanol followed by overnight extraction in methanol at 60 degrees C. The methanol extract was evaporated to dryness, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture [methadone-d9 and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)-d3] and 0.1M HCI were added to approximately 20 mg of specimen or spiked blank hair and sonicated for 1 h. The pH was adjusted to neutral, and methadone and its primary metabolite, EDDP, were analyzed by GC-MS following solid-phase extraction using Bond Elute Certify columns and pH 7.4 phosphate buffer (0.1M). Forty hair specimens were confirmed positive for methadone by GC-MS. Concentrations ranged from 0.10 to 8.3 ng/mg for methadone and 0.1 to 1.2 ng/mg for EDDP. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results with a cutoff of 0.1 ng/mg for both methadone and EDDP as the reference method. The optimum cutoff for the Cozart Methadone Microplate ELISA was determined to be between 200 and 300 pg methadone equivalents/mg hair using a 20 mg hair sample. The Cozart Methadone Microplate EIA for methadone and metabolites in hair using a cut-off of 200 pg/mg hair with a 20 mg hair sample had a sensitivity of 95 +/- 2% and a specificity of 100 +/- 3.5% (vs GC-MS) and an accuracy of 98.2 +/- 1.3%.

The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.

The purpose of these studies was to determine the performance characteristics of the Cozart microplate EIA for the detection of cocaine and cocaine metabolites in oral fluid. A total of 217 samples were collected using the Cozart RapiScan oral fluid Collection System from donors who were receiving treatment and being monitored for their addiction. The samples were screened in the laboratory using the Cozart microplate EIA for cocaine and metabolites and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. Of the 217 samples tested, 116 were positive for cocaine and/or cocaine metabolites. The Cozart microplate EIA for cocaine, using a cutoff of 30 ng/mL benzoylecgonine equivalents in neat oral fluid, had a sensitivity of 95.7% +/- 2.0% and specificity of 100% +/- 0.7%. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

The purpose of this study was to evaluate the efficiency of the Cozart RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen at -20 degrees C until analysis by GC-MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Oral fluid has become a widely accepted alternative matrix for drugs of abuse detection. Immunoassays have been developed for on-site testing of cocaine and metabolites in oral fluid. The performance of the Cozart RapiScan Oral Fluid Drug Testing System (CRS) was evaluated in comparison with Cozart Microplate Enzyme Immunoassay Cocaine Oral Fluid Kit (COC ELISA) and gas chromatography-mass spectrometry (GC-MS) at several screening and confirmation cutoffs, including those proposed by SAMHSA and those currently in use in the U.K. Oral fluid samples (n = 1271) were collected prior to and following controlled clinical cocaine administration. CRS provides a qualitative screen at a preset cutoff of 30 microg/L. Sensitivity, specificity, and efficiency for CRS (30 microg/L) as compared with COC ELISA with a cutoff of 30 microg/L were 92.1%, 91.8%, and 92.0%. The comparison of CRS (30 microg/L) with the 8-mg/L proposed SAMHSA confirmation cutoffs for cocaine and/or benzoylecgonine exhibited a sensitivity of 82.7%, a specificity of 94.5%, and an efficiency of 87.6%. For this study, an alternative CRS cutoff of 20 microg/L was also evaluated. Performance characteristics of CRS (20 microg/L) at the proposed SAMHSA confirmation cutoffs were 89.9%, 89.7%, and 89.8%, respectively. At cutoffs in use in the U.K., 30- micro g/L CRS screen and 15- microg/L GC-MS cutoffs for cocaine, benzoylecgonine, and/or ecgonine methyl ester sensitivity, specificity, and efficiency were 89.4%, 92.2%, and 90.7%, respectively. Cozart RapiScan had performance similar to the COC ELISA assay for the detection of cocaine exposure and suitable sensitivity and specificity at the proposed SAMHSA cutoffs.

Oral fluid specimens (N = 1406) were collected from 19 subjects prior to and up to 72 h following controlled administration of oral codeine. Volunteers provided informed consent to participate in this National Institute on Drug Abuse Institutional Review Board-approved protocol. A modification of Cozart Microplate Opiate EIA Oral Fluid Kit (Opiate ELISA), employing codeine calibrators, was used for semiquantitative analysis of opiates, followed by gas chromatography-mass spectrometry (GC-MS) for the confirmation and quantitation of codeine, norcodeine, morphine, and normorphine in oral fluid. GC-MS limits of detection and quantitation were 2.5 microg/L for all analytes. The Substance Abuse and Mental Health Services Administration (SAMHSA) has proposed a 40-microg/L opiate screening and a 40-microg/L morphine or codeine confirmation cutoff for the detection of opiate use. Oral fluid opiate screening and confirmation cutoffs of 30 micro g/L are in use in the U.K. Utilizing 2.5-, 20-, 30-, and 40-microg/L GC-MS cutoffs, 26%, 20%, 19%, and 18% of the oral fluid specimens were positive for codeine or one of its metabolites. Six Opiate ELISA/confirmation cutoff criteria (2.5/2.5, 10/2.5, 20/20, 30/20, 30/30, and 40/40 microg/L) were evaluated. Calculations for Opiate ELISA sensitivity, specificity, and efficiency were determined from the number of true-positive, true-negative, false-positive, and false-negative results at each screening/confirmation cutoff. Sensitivity, specificity, and efficiency for the lowest cutoff were 91.5%, 88.6%, and 89.3%. Application of the cutoff currently used in the U.K. yielded sensitivity, specificity, and efficiency results of 79.7%, 99.0%, and 95.4% and similar results of 76.7%, 99.1%, and 95.1% when applying the SAMHSA criteria. These data indicate that the Opiate ELISA efficiently detects oral codeine use. In addition, the data, collected following controlled oral codeine administration, may aid in the interpretation of opiate oral fluid test results and in the selection of appropriate oral fluid screening and confirmation cutoffs.

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics91% when compared to GC/MS at the proposed SAMHSA cutoff.

Two experiments are described in this paper, which examine the processing of English sentences containing “complement” verbs, and which may be followed either by a nounphrase, as a direct object, or by a complement clause. It has been claimed by Frazier and Rayner (1982) that subjects are “garden-pathed” when reading reduced complements (lacking the overt complemetiser), and that this fact is strong evidence in support of the application of the principle of “Minimal Attachment” as a universal property of the human parser. Holmes, Kennedy, and Murray (1987) questioned this conclusion by providing evidence that full and reduced complements present equivalent problems, probably because of their greater structural complexity. Rayner and Frazier (1987) disputed this conclusion, ascribing it to an artefact resulting from the use of a self-paced reading task. The first experiment examines this controversy with a replication of the study by Holmes et al., measuring eye movements as the sentences are proc...