Michael T. Kelliher

Background: Immunoassay drug screens provide rapid analysis of urine for the presence of therapeutics and drugs of abuse. Compared to definitive (confirmatory) methods, immunoassays are prone to false-positive and -negative results. Laboratories generally rely on manufacturers’ claims regarding method sensitivity and specificity; few have the resources to independently verify performance. In this study, we review the performance of our opioid immunoassay drug screens in comparison to a definitive method. Methods: Results of 859 urine samples tested via opioid immunoassay screens for buprenorphine, fentanyl, methadone metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EDDP), opiates, and oxycodone were compared to definitive results obtained via liquid chromatography–tandem mass spectrometry (LC-MS/MS). The data examined here included multiple samples from individual patients. Our quantitative LC-MS/MS method includes 19 opioid compounds (parent drugs plus metabolites). Results: Immunoassay sensitivity and specificity ranged from 96% to 100% and 84% to 99%, respectively. The sensitivity and specificity of these screens were similar to manufacturers’ claims with some exceptions. The opiates immunoassay had poor performance when limiting the comparison to its target compound, morphine, but improved when including all compounds listed in the manufacturer’s instructions for use (IFU). While demonstrating good sensitivity, the buprenorphine immunoassay demonstrated lower specificity than stated in the IFU. Conclusions: The opioid immunoassay screens in use at our facility compared favorably to a definitive LC-MS/MS method. The urine fentanyl screen had the lowest sensitivity (96%) and had a specificity of 97%. The urine buprenorphine assay was the least specific (84%) and had a sensitivity of 99%.

Background: The aim of this study was to redefine the icterus index cutoff for the Roche Jaffé creatinine method using both conjugated and unconjugated bilirubin on 3 Roche cobas modules (c311, c501, and c701/c702) at laboratories across our hospital network. Methods: Interference was evaluated by adding conjugated bilirubin (as bilirubin conjugate, ditaurate) and unconjugated bilirubin to pooled remnant plasma. The effects of conjugated and unconjugated bilirubin were tested separately to assess the contribution of each species. The magnitude of interference was calculated as both absolute and percentage error with total allowable error limits set at 0.1 mg/dL (8 µmol/L or 8%). Results: Analysis of interference data across the 3 Roche modules did not show bias exceeding our total allowable error limits for plasma creatinine up to a conjugated bilirubin icterus index of 16.2 (approximately 16.2 mg/dL or 277 μmol/L) or an unconjugated bilirubin icterus index of 18.5 (approximately 18.5 mg/dL or 316 μmol/L), the highest concentrations tested. Conclusions: Our results demonstrate that the Roche Jaffé method exhibits acceptable performance in the presence of icterus at icterus indexes above the manufacturer’s current recommendations of 5 (approximately 5 mg/dL or 86 μmol/L) and 10 (approximately 10 mg/dL or 171 μmol/L) for conjugated and unconjugated bilirubin, respectively. We have updated the icterus index in our hospital system to 16 for conjugated bilirubin and 18 for unconjugated bilirubin.

Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure–function analysis of endogenous Drosophila melanogaster kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.

Significance: We previously demonstrated that the microtubule (MT) motor protein kinesin-1 slides and transports MTs themselves in addition to its established role in organelle transport. However, the physiological importance of MT sliding has not been determined. Here, we identified the mechanism of kinesin-1–based MT sliding and generated a sliding-deficient Drosophila mutant. Additionally, we generated a chimeric motor that actively slides MTs but cannot transport organelles. Using these tools, we demonstrated that MT sliding is an essential biological process, with key roles in both axon and dendrite outgrowth. Because the C-terminal MT-binding site of kinesin-1, which is essential for sliding, is highly conserved in vertebrates and invertebrates, we postulate that MT sliding is important for nervous system development in many organisms.

Novel copolymers of trisubstituted ethylene monomers, ring-substituted 2-phenyl-1,1-dicyanoethylenes, RC₆H₃CH˭C(CN)₂ (where R is 2,3-(CH₃O)₂, 2,4-(CH₃O)₂, 2,5-(CH₃O)₂, 2,6-(CH₃O)₂, 3,4-(CH₃O)₂, and 3,5-(CH₃O)₂ and 4-fluorostyrene were prepared at equimolar monomer feed composition by solution copolymerization in the presence of a radical initiator (ABCN) at 70°C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, ¹H and ¹³C-NMR. The order of relative reactivity (1/r ₁) for the monomers is 2,6-(CH₃O)₂(2.8) > 2,5-(CH₃O)₂(2.5) > 2,3-(CH₃O)₂ (2.1) > 3,5-(CH₃O)₂ (1.8) > 3,4-(CH₃O)₂ (0.9) > 2,4-(CH₃O)₂ (0.7). High T_g of the copolymers, in comparison with that of poly(4-fluorostyrene) indicates a substantial decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in the 250–400°C range with residue, which then decomposition in 400–800°C range.