Metabolism of methylstenbolone studied with human liver microsomes and the uPA+/+‐SCID chimeric mouse model

Anti‐doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono‐hydroxylated derivatives (U1–U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di‐hydroxylated metabolites (U11–U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC‐MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. Copyright © 2014 John Wiley & Sons, Ltd.     

Analytical study of the Middle Kingdom group of gold jewellery from tomb 124 at Riqqa,Egypt

The jewellery from tomb 124 at Riqqa, consisting of one pectoral and one winged beetle in gold and cloisonné work, one gold shell pendant decorated with wires and granulation, and one hollow gold amulet in the form of god Min, was analysed by handheld X-ray fluorescence and scanning electron microscopy with energy-dispersive X-ray spectroscopy. This group of jewellery, dated to the second half of the 12th Dynasty (c. 1900–1840 B.C.), was excavated inside the coffin of an adult male, which had been crushed after burial by the collapse of the chamber roof during an episode of looting. Both the male and the looter's body were found inside the chamber, evidencing that the group of jewellery was intact. Despite having been highly restored in the past, as referenced in the correspondence between the excavator Flinders Petrie and the curators of the Manchester Museum, it could be shown that the jewellery was produced using Ag-rich electrum alloys containing platinum group element inclusions that indicate the use of alluvial gold. The analysis of some joins has confirmed the use of hard-soldering, with solders obtained by addition of Cu to the base-alloy. Data obtained for the jewellery of tomb 124 were compared with data previously obtained for tomb 296, also excavated at Riqqa, but dated to the 18th Dynasty. The comparison demonstrates the continuity of the workshop traditions in one location between the Middle Kingdom and the New Kingdom but also reveals discrepancies in the alloys employed in those two periods.     

Inter-laboratory performance between two nanoparticle air filtration systems using scanning mobility particle analyzers

The performance of two aerosol testing systems, at two different laboratories (University of Nebraska Medical Center—UNMC and 3M Company), was compared to evaluate which calibration procedures minimized variability in filter testing of nanoparticles. Both charged electret and uncharged flat-web fibrous filters were used with Scanning Mobility Particle Sizers to give upstream and downstream size distributions and calculate filter penetration. Challenge aerosols were polydisperse nanoparticles of sodium chloride (NaCl) ranging from approximately 10–300 nm and monodisperse polystyrene latex (PSL) spheres of preselected sizes, including 40, 60, 100, and 200 nm. The implementation of optimized procedures resulted in comparable filtration performance at the two testing sites with challenges of NaCl particles and PSL spheres. The penetration results for the uncharged filter were nearly identical for both challenges, while lower penetration through the charged filter was observed with NaCl aerosol, probably due to differences in NaCl and PSL dielectric constants. Results showed that reproducible, comparable nanoparticle filtration data could be achieved between two separate laboratories when sources of error and proper calibration procedures were addressed.     

Time to default in credit scoring using survival analysis: a benchmark study

We investigate the performance of various survival analysis techniques applied to ten actual credit data sets from Belgian and UK financial institutions. In the comparison we consider classical survival analysis techniques, namely the accelerated failure time models and Cox proportional hazards regression models, as well as Cox proportional hazards regression models with splines in the hazard function. Mixture cure models for single and multiple events were more recently introduced in the credit risk context. The performance of these models is evaluated using both a statistical evaluation and an economic approach through the use of annuity theory. It is found that spline-based methods and the single event mixture cure model perform well in the credit risk context.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Structural and immunochemical analysis of the lipopolysaccharide from Acinetobacter lwoffii F78 located outside Chlamydiaceae with a Chlamydia-specific lipopolysaccharide epitope

Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.     

In vitro and in vivo metabolism studies of dimethazine

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti‐doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA^+/+‐SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called ‘Xtreme DMZ’. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC‐High Resolution Mass Spectrometry (HRMS) and GC‐MS(/MS) analysis methasterone and six other dimethazine metabolites (M1–M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro‐conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.     

An Akaike information criterion for multiple event mixture cure models

We derive the proper form of the Akaike information criterion for variable selection for mixture cure models, which are often fit via the expectation–maximization algorithm. Separate covariate sets may be used in the mixture components. The selection criteria are applicable to survival models for right-censored data with multiple competing risks and allow for the presence of a non-susceptible group. The method is illustrated on credit loan data, with pre-payment and default as events and maturity as the non-susceptible case and is used in a simulation study.