A simplified procedure for GC/C/IRMS analysis of underivatized 19-norandrosterone in urine following HPLC purification

Nandrolone and/or its precursors are included in the World Anti-doping Agency (WADA) list of forbidden substances and methods and as such their use is banned in sport. 19-Norandrosterone (19-NA) the main metabolite of these compounds can also be produced endogenously. The need to establish the origin of 19-NA in human urine samples obliges the antidoping laboratories to use isotope ratio mass spectrometry (IRMS) coupled to gas chromatography (GC/C/IRMS). In this work a simple liquid chromatographic method without any additional derivatization step is proposed, allowing to drastically simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis. The purity of the extracts was verified by parallel analysis by gas chromatography coupled to mass spectrometry with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision of ±0.3‰ and between assay precision of ±0.4‰). The method has been tested with samples obtained after the administration of synthetic 19-norandrostenediol and samples collected during pregnancy where 19-NA is known to be produced endogenously. Twelve drugs and synthetic standards able to produce through metabolism 19-NA have shown to present δ¹³C values around −29‰ being quite homogeneous (−28.8 ± 1.5; mean ± standard deviation) while endogenously produced 19-NA has shown values comparable to other endogenous produced steroids in the range −21 to −24‰ as already reported. The efficacy of the method was tested on real samples from routine antidoping analyses.     

Lipase maturation factor 1 is required for endothelial lipase activity

Lipase maturation factor 1 (Lmf1) is an endoplasmic reticulum (ER) membrane protein involved in the posttranslational folding and/or assembly of lipoprotein lipase (LPL) and hepatic lipase (HL) into active enzymes. Mutations in Lmf1 are associated with diminished LPL and HL activities ("combined lipase deficiency") and result in severe hypertriglyceridemia in mice as well as in human subjects. Here, we investigate whether endothelial lipase (EL) also requires Lmf1 to attain enzymatic activity. We demonstrate that cells harboring a (cld) loss-of-function mutation in the Lmf1 gene are unable to generate active EL, but they regain this capacity after reconstitution with the Lmf1 wild type. Furthermore, we show that cellular EL copurifies with Lmf1, indicating their physical interaction in the ER. Finally, we determined that post-heparin phospholipase activity in a patient with the LMF1(W464X) mutation is reduced by more than 95% compared with that in controls. Thus, our study indicates that EL is critically dependent on Lmf1 for its maturation in the ER and demonstrates that Lmf1 is a required factor for all three vascular lipases, LPL, HL, and EL.     

Mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC(2)(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.     

Comparative analysis of methods for estimating arm segment parameters and joint torques from inverse dynamics

A common problem in the analyses of upper limb unfettered reaching movements is the estimation of joint torques using inverse dynamics. The inaccuracy in the estimation of joint torques can be caused by the inaccuracy in the acquisition of kinematic variables, body segment parameters (BSPs), and approximation in the biomechanical models. The effect of uncertainty in the estimation of body segment parameters can be especially important in the analysis of movements with high acceleration. A sensitivity analysis was performed to assess the relevance of different sources of inaccuracy in inverse dynamics analysis of a planar arm movement. Eight regression models and one water immersion method for the estimation of BSPs were used to quantify the influence of inertial models on the calculation of joint torques during numerical analysis of unfettered forward arm reaching movements. Thirteen subjects performed 72 forward planar reaches between two targets located on the horizontal plane and aligned with the median plane. Using a planar, double link model for the arm with a floating shoulder, we calculated the normalized joint torque peak and a normalized root mean square (rms) of torque at the shoulder and elbow joints. Statistical analyses quantified the influence of different BSP models on the kinetic variable variance for given uncertainty on the estimation of joint kinematics and biomechanical modeling errors. Our analysis revealed that the choice of BSP estimation method had a particular influence on the normalized rms of joint torques. Moreover, the normalization of kinetic variables to BSPs for a comparison among subjects showed that the interaction between the BSP estimation method and the subject specific somatotype and movement kinematics was a significant source of variance in the kinetic variables. The normalized joint torque peak and the normalized root mean square of joint torque represented valuable parameters to compare the effect of BSP estimation methods on the variance in the population of kinetic variables calculated across a group of subjects with different body types. We found that the variance of the arm segment parameter estimation had more influence on the calculated joint torques than the variance of the kinematics variables. This is due to the low moments of inertia of the upper limb, especially when compared with the leg. Therefore, the results of the inverse dynamics of arm movements are influenced by the choice of BSP estimation method to a greater extent than the results of gait analysis.     

Colloidal properties of biodegradable nanoparticles influence interaction with amyloid-β peptide

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of amyloid-β peptides (Aβ). During the past few years, promising approaches based on nanotechnologies have emerged to alter Aβ aggregation and its related toxicity. This study aims to investigate the influence of the nanoparticle colloidal properties over the interaction with Aβ peptide 1–42 (Aβ_1–42). Using capillary electrophoresis with laser-induced fluorescence detection, it was shown that biodegradable poly(ethylene glycol)-block-polylactide (PEG-b-PLA) nanoparticles were able to interact with Aβ_1–42 peptide leading to its uptake in rather short time periods. In addition, we highlighted the crucial role of the nanocarrier colloidal properties on the uptake kinetics. Whereas nanoparticles stabilized by sodium cholate (lower size and higher negative surface charge) gave optimum uptake kinetics, nanoparticles stabilized with others surfactants presented lower interactions. In contrast, PEG density seemed to have no influence on the interaction when sodium cholate was used for the preparation. This study intends to give new insights into Aβ_1–42 peptide interaction with nanoparticulate systems by helping to determine suitable nanoparticle characteristics regarding forthcoming therapeutic strategies against AD.