Validation of a liquid chromatography-tandem mass spectrometry method for analyzing cannabinoids in oral fluid.

A liquid chromatography tandem mass spectrometry method was developed for quantifying ten cannabinoids in oral fluid (OF). This method utilizes OF collected by the Quantisal™ device and concurrently quantifies cannabinol (CBN), cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-THC (11-OH-THC), 11-nor-9-carboxy-Δ9-THC (THC-COOH), 11-nor-9-carboxy-Δ9-THC glucuronide (THC-COOH-gluc), Δ9-THC glucuronide (THC-gluc), cannabigerol (CBG), tetrahydrocannabiverin (THCV), and Δ9-tetrahydrocannabinolic acid A (THCA-A). Solid phase extraction was optimized using Oasis Prime HLB 30 mg 96-well plates. Cannabinoids were separated by liquid chromatography over a BEH C18 column and detected by a Waters TQ-S micro tandem mass spectrometer. The lower limits of quantification (LLOQ) were 0.4 ng/mL for CBN, CBD, THC, 11-OH-THC, THC-gluc, and THCV; and 1.0 ng/mL for THC-COOH, THC-COOH-gluc, CBG and THCA-A. Linear ranges extended to 2000 ng/mL for THC and 200 ng/mL for all other analytes. Inter-day analytical bias and imprecision at three levels of quality control (QC) was within ±15%. Mean extraction efficiencies ranged from 26.0-98.8%. Applicability of this method was tested using samples collected from individuals randomly assigned to smoke either a joint containing <0.1%, 5.9%, or 13.4% THC content. This method was able to identify and calculate the concentration of 6 of 10 cannabinoids validated in this method

Frailty of Māori, Pasifika, and non-Māori/non-Pasifika older people in New Zealand: a national population study of older people referred for home care services

Little is known about the prevalence of frailty in indigenous populations. We developed a frailty index for older New Zealand Māori and Pasifika who require publicly funded support services.A frailty index (FI) was developed for New Zealand adults aged ≥65 years who had an interRAI-Home Care assessment between 1 June 2012 and 30 October 2015. A frailty score for each participant was calculated by summing the number of deficits recorded and dividing by the total number of possible deficits. This created a FI with a potential range from 0 to 1. Linear regression models for FIs with ethnicity were adjusted for age and sex. Cox proportional hazards models were used to assess the association between the FI and mortality for Māori, Pasifika, and non-Māori/non-Pasifika.Of 54,345 participants, 3,096 (5.7%) identified as Māori, 1,846 (3.4%) were Pasifika, and 49,415 (86.7%) identified as neither Māori nor Pasifika. New Zealand Europeans (48,178, 97.5%) constituted most of the latter group. Within each sex, the mean FIs for Māori and Pasifika were greater than the mean FIs for non-Māori and non-Pasifika, with the difference being more pronounced in females. The FI was associated with mortality (Māori SHR 2.53, 95% CI 1.63 to 3.95; Pasifika SHR 6.03, 95% CI 3.06 to 11.90; non-Māori and non-Pasifika SHR 2.86, 95% 2.53 to 3.25).This study demonstrated differences in FI between the ethnicities in this select cohort. After adjustment for age and sex, increases in FI were associated with increased mortality. This suggests that FI is predictive of poor outcomes in these ethnic groups

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. Reliable and accurate quantification of the proteins present in a cell or tissue remains a major challenge for post-genome scientists. Proteins are the primary functional molecules in biological systems and knowledge of their abundance and dynamics is an important prerequisite to a complete understanding of natural physiological processes, or dysfunction in disease. Accordingly, much effort has been spent in the development of reliable, accurate and sensitive techniques to quantify the cellular proteome, the complement of proteins expressed at a given time under defined conditions (1). Moreover, the ability to model a biological system and thus characterize it in kinetic terms, requires that protein concentrations be defined in absolute numbers (2, 3). Given the high demand for accurate quantitative proteome data sets, there has been a continual drive to develop methodology to accomplish this, typically using mass spectrometry (MS) as the analytical platform. Many recent studies have highlighted the capabilities of MS to provide good coverage of the proteome at high sensitivity often using yeast as a demonstrator system (4⇓⇓⇓⇓⇓–10), suggesting that quantitative proteomics has now “come of age” (1). However, given that MS is not inherently quantitative, most of the approaches produce relative quantitation and do not typically measure the absolute concentrations of individual molecular species by direct means. For the yeast proteome, epitope tagging studies using green fluorescent protein or tandem affinity purification tags provides an alternative to MS. Here, collections of modified strains are generated that incorporate a detectable, and therefore quantifiable, tag that supports immunoblotting or fluorescence techniques (11, 12). However, such strategies for copies per cell (cpc) quantification rely on genetic manipulation of the host organism and hence do not quantify endogenous, unmodified protein. Similarly, the tagging can alter protein levels - in some instances hindering protein expression completely (11). Even so, epitope tagging methods have been of value to the community, yielding high coverage quantitative data sets for the majority of the yeast proteome (11, 12). MS-based methods do not rely on such nonendogenous labels, and can reach genome-wide levels of coverage. Accurate estimation of absolute concentrations i.e. protein copy number per cell, also usually necessitates the use of (one or more) external or internal standards from which to derive absolute abundance (4). Examples include a comprehensive quantification of the Leptospira interrogans proteome that used a 19 protein subset quantified using selected reaction monitoring (SRM)1 to calibrate their label-free data (8, 13). It is worth noting that epitope tagging methods, although also absolute, rely on a very limited set of standards for the quantitative western blots and necessitate incorporation of a suitable immunogenic tag (11). Other recent, innovative approaches exploiting total ion signal and internal scaling to estimate protein cellular abundance (10, 14), avoid the use of internal standards, though they do rely on targeted proteomic data to validate their approach. The use of targeted SRM strategies to derive proteomic calibration standards highlights its advantages in comparison to label-free in terms of accuracy, precision, dynamic range and limit of detection and has gained currency for its reliability and sensitivity (3, 15⇓–17). Indeed, SRM is often referred to as the “gold standard proteomic quantification method,” being particularly well-suited when the proteins to be quantified are known, when appropriate surrogate peptides for protein quantification can be selected a priori, and matched with stable isotope-labeled (SIL) standards (18⇓–20). In combination with SIL peptide standards that can be generated through a variety of means (3, 15), SRM can be used to quantify low copy number proteins, reaching down to ∼50 cpc in yeast (5). However, although SRM methodology has been used extensively for S. cerevisiae protein quantification by us and others (19, 21, 22), it has not been used for large protein cohorts because of the requirement to generate the large numbers of attendant SIL peptide standards; the largest published data set is only for a few tens of proteins. It remains a challenge therefore to robustly quantify an entire eukaryotic proteome in absolute terms by direct means using targeted MS and this is the focus of our present study, the Census Of the Proteome of Yeast (CoPY). We present here direct and absolute quantification of nearly 2000 endogenous proteins from S. cerevisiae grown in steady state in a chemostat culture, using the SRM-based QconCAT approach. Although arguably not quantification of the entire proteome, this represents an accurate and rigorous collection of direct yeast protein quantifications, providing a gold-standard data set of endogenous protein levels for future reference and comparative studies. The highly reproducible SIL-SRM MS data, with robust CVs typically less than 20%, is compared with other extant data sets that were obtained via alternative analytical strategies. We also report a matched high quality transcriptome from the same cells using RNA-seq, which supports additional calculations including a refined estimate of the total protein content in yeast cells, and a simple linear model of translation explaining 70% of the variance between RNA and protein levels in yeast chemostat cultures. These analyses confirm the validity of our data and approach, which we believe represents a state-of-the-art absolute quantification compendium of a significant proportion of a model eukaryotic proteome

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 μg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving

Economies of (Alleged) Deviance: Sex Work and the Sport Mega Event

Based on ethnographic data collected during the 2014 FIFA World Cup and 2016 Olympic Games in Rio de Janeiro, Brazil, thisarticle is interested to examine urban processes which reinvent the changing (sexual) landscape. Focusing on the way (host) citiesshape sex work both imaginatively and physically, we explore the (lived) realities of neoliberal imaginaries that shape urbanspace. Often thought to exist in the urban shadow as an absent-presence in cosmopolitan processes, we demonstrate the manner inwhich sexualized and racialized women creatively resist the political and economic trajectories of neoliberal urbanism that seek toexpropriate land and dispossess certain bodies. In the context of Rio de Janeiro—as in other host cities—this is particularlyevident in the routine encounter between sexual minorities and local law enforcement. Mindful of the literature on state incursioninto social-sexual life, we remain attentive to the everyday strategies through which those deemed sexually deviant and/or victimnavigate local authorities in search of new opportunities for economic salvation in the midst of the sport mega-event

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10822-011-9454-9) contains supplementary material, which is available to authorized users

A self-controlled case series to assess the effectiveness of beta blockers for heart failure in reducing hospitalisations in the elderly

Background: To determine the suitability of using the self-controlled case series design to assess improvements in health outcomes using the effectiveness of beta blockers for heart failure in reducing hospitalisations as the example. Methods: The Australian Government Department of Veterans' Affairs administrative claims database was used to undertake a self-controlled case-series in elderly patients aged 65 years or over to compare the risk of a heart failure hospitalisation during periods of being exposed and unexposed to a beta blocker. Two studies, the first using a one year period and the second using a four year period were undertaken to determine if the estimates varied due to changes in severity of heart failure over time. Results: In the one year period, 3,450 patients and in the four year period, 12, 682 patients had at least one hospitalisation for heart failure. The one year period showed a non-significant decrease in hospitalisations for heart failure 4-8 months after starting beta-blockers, (RR, 0.76; 95% CI (0.57-1.02)) and a significant decrease in the 8-12 months post-initiation of a beta blocker for heart failure (RR, 0.62; 95% CI (0.39, 0.99)). For the four year study there was an increased risk of hospitalisation less than eight months post-initiation and significant but smaller decrease in the 8-12 month window (RR, 0.90; 95% CI (0.82, 0.98)). Conclusions: The results of the one year observation period are similar to those observed in randomised clinical trials indicating that the self-controlled case-series method can be successfully applied to assess health outcomes. However, the result appears sensitive to the study periods used and further research to understand the appropriate applications of this method in pharmacoepidemiology is still required. The results also illustrate the benefits of extending beta blocker utilisation to the older age group of heart failure patients in which their use is common but the evidence is sparse.Emmae N Ramsay, Elizabeth E Roughead, Ben Ewald, Nicole L Pratt and Philip Rya

Horizontal Branch Stars: The Interplay between Observations and Theory, and Insights into the Formation of the Galaxy

We review HB stars in a broad astrophysical context, including both variable and non-variable stars. A reassessment of the Oosterhoff dichotomy is presented, which provides unprecedented detail regarding its origin and systematics. We show that the Oosterhoff dichotomy and the distribution of globular clusters (GCs) in the HB morphology-metallicity plane both exclude, with high statistical significance, the possibility that the Galactic halo may have formed from the accretion of dwarf galaxies resembling present-day Milky Way satellites such as Fornax, Sagittarius, and the LMC. A rediscussion of the second-parameter problem is presented. A technique is proposed to estimate the HB types of extragalactic GCs on the basis of integrated far-UV photometry. The relationship between the absolute V magnitude of the HB at the RR Lyrae level and metallicity, as obtained on the basis of trigonometric parallax measurements for the star RR Lyrae, is also revisited, giving a distance modulus to the LMC of (m-M)_0 = 18.44+/-0.11. RR Lyrae period change rates are studied. Finally, the conductive opacities used in evolutionary calculations of low-mass stars are investigated. [ABRIDGED]Comment: 56 pages, 22 figures. Invited review, to appear in Astrophysics and Space Scienc