A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps

BACKGROUND: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. RESULTS: A total of 110,592 maize BAC clones (~ 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize [1,2]. CONCLUSION: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Urine drug testing of chronic pain patients. II. Prevalence patterns of prescription opiates and metabolites

This study of 20,089 urine specimens from chronic pain patients provided a unique opportunity to evaluate the prevalence of prescription opiates and metabolites, assess the usefulness of inclusion of normetabolites in the test panel, and compare opiate and oxycodone screening results to liquid chromatography with tandem mass spectrometry (LC-MS-MS) results. All specimens were screened by an opiate [enzyme-linked immunosorbent assay (ELISA), 100 ng/mL] and oxycodone assay [ELISA, 100 ng/mL or enzyme immunoassay (EIA), 50 ng/mL] and simultaneously tested by LC-MS-MS [limit of quantitation (LOQ) = 50 ng/mL] for 10 opiate analytes (codeine, norcodeine, morphine, hydrocodone, dihydrocodeine, norhydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone). Approximately two-thirds of the specimens were positive for one or more opiate analytes. The number of analytes detected in each specimen varied from 1 to 8 with 3 (34.8%) being most prevalent. Hydrocodone and oxycodone (in combination with metabolites) were most prevalent followed by morphine. Norcodeine was only infrequently detected whereas the prevalence of norhydrocodone and noroxycodone was approximately equal to the prevalence of the parent drug. A substantial number of specimens were identified that contained norhydrocodone (n = 943) or noroxycodone (n = 702) but not the parent drug, thereby establishing their interpretative value as biomarkers of parent drug use. Comparison of the two oxycodone screening assays revealed that the oxycodone ELISA had broader cross-reactivity with opiate analytes, and the oxycodone EIA was more specific for oxycodone. Specimens containing only norhydrocodone were best detected with the opiate ELISA whereas noroxycodone (only) specimens were best detected by the oxycodone EIA. Introduction Opioids are the mainstay of pharmacotherapy in patients suffering from acute and chronic pain. Opioid drugs are generally safe and efficacious when used as prescribed but also have powerful reinforcing properties and high abuse potential for some individuals. Clinicians who prescribe opioids are concerned about the risks of under-treatment, over-prescription, and the possibility of opioid addiction (1). Drug testing of chronic pain patients maintained on opioids provides objective information about recent use of prescribed and unauthorized drugs and illicit drugs. For patients prescribed one or more of the structurally related "opiate" drugs, interpretation of their test results can be difficult because of the multiple metabolic pathways accorded these compounds in the human body prior to their excretion. Interpretation is especially difficult for those drugs that are biotransformed into other commercially available prescription opiates. For example, two of the most frequently prescribed analgesics, hydrocodone and oxycodone, undergo O-demethylation to hydromorphone and oxymorphone, respectively. In addition, hydrocodone undergoes 6-keto-reduction to dihydrocodeine (6-α-hydrocodo

Caffeine content of specialty coffees.

Abstract I caffeine is the world's most widely consumed drug with its main source found in coffee. We evaluated the caffeine content of caffeinated and decaffeinated specialty coffee samples obtained from coffee shops. Caffeine was isolated from the coffee by liquid-liquid extraction and analyzed by gas chromatography with nitrogen-phosphorus detection. In this study, the coffees sold as decaffeinated were found to have caffeine concentrations less than 17.7 rag/dose. There was a wide range in caffeine content present in caffeinated coffees ranging from 58 to 259 rag/dose. The mean (SD) caffeine content of the brewed specialty coffees was 188 (36) mg for a 16-oz cup. Another notable find is the wide range of caffeine concentrations (259-564 rag/dose) in the same coffee beverage obtained from the same outlet on six consecutive days

Intravenous and Oral l-␣-Acetylmethadol: Pharmacodynamics and Pharmacokinetics in Humans 1

ABSTRACT Levo-␣-acetylmethadol (LAAM) is a long-acting opioid agonist approved for use as a maintenance treatment for opioid dependence. Previous clinical studies report that the onset of the effects of LAAM is slower after parenteral administration than oral administration; however, preclinical studies suggest otherwise. This study examined the pharmacodynamic and pharmacokinetic profile of LAAM when given orally and intravenously to humans. Opioid-experienced volunteers (n ϭ 6), who were not physically dependent on opioids, received LAAM (20 and 40 mg/70 kg i.v. and p.o.) and placebo under double-blind, double-dummy conditions during five weekly experimental sessions. Behavioral, physiological, subjective and pharmacokinetic measures were collected before and for 96 hr after drug administration. Intravenous LAAM produced significant subjective and physiological effects that appeared within 5 min, whereas the effects of oral LAAM appeared more slowly within 1 to 2 hr after drug administration. Pharmacokinetic data indicate that the immediate effects of intravenous LAAM are largely attributable to the parent drug rather than the active metabolites, nor-LAAM and dinor-LAAM. LAAM produced prototypic opioid agonist effects (i.e., miosis, subjective ratings of high, nodding) that were of equal magnitude across routes, doserelated and of long duration (up to 60 hr). These data are in contrast to previous clinical reports and indicate that LAAM produces effects of immediate onset when administered parenterally, which suggests that intravenous LAAM possesses greater abuse potential than previously believed. Levo-␣-acetylmethadol is a synthetic opioid that was approved for use as a treatment for opioid dependence by the Food and Drug Administration in 1993. A congener of methadone, LAAM produces opioid effects typical of mu agonists, including analgesia, euphoria, miosis and respiratory depression The persistent action of LAAM has been attributed primarily to its sequential N-demethylation to two primary active metabolites, nor-LAAM and dinor-LAA