Carbon isotope ratio (δC) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (δ¹³C) profiling study (n = 1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This δ¹³C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to δ¹³C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n = 100; consisting of three countries) is also presented with comparison to δ¹³C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.     

Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies

Monoclonal antibodies are the fastest growing class of biologics in the pharmaceutical industry. The correlation between mAb glycosylation and aggregation has not been elucidated in detail, yet understanding the structure-stability relationship involving glycosylation is critical for developing successful drug formulations. We conducted studies of temperature-induced aggregation and compared the stability of both glycosylated and aglycosylated forms of a human IgG1. In parallel, we also performed molecular dynamics simulations of the glycosylated full antibody to gain an understanding of the polysaccharide surroundings at the molecular level. Aglycosylated mAbs are somewhat less stable and therefore aggregate more easily than the glycosylated form at the temperatures studied. Glycosylation seems to enhance solubility and stability of these therapeutics and thus might be important for long-term storage.     

Metronidazole induces gametocytogenesis in gregarine associations maintained in vitro

Gametocytogenesis was induced in mature associations of Protomagalhaensia wolfi and Protomagalhaensia blaberae maintained in vitro by inclusion of metronidazole in the culture medium. The response was neither strictly dosage dependent nor uniform across gregarine species. We hypothesize that metronidazole induces gregarine gametocytogenesis by disrupting PUF2 proteins responsible for the translational control of sexual development and gametocytogenesis in apicomplexans.     

Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.     

MORPHOLOGICAL,ANATOMICAL, AND MOLECULAR CONSEQUENCES OF GROWTH AND DEVELOPMENT AT LOW TEMPERATURE IN SECALE CEREALE

Growth of winter cereals at low, nonfreezing temperatures is essential for the establishment of a cold-hardy state and subsequent survival of the overwintering plant. In this paper, I describe the consequences of growth and development of Secale cereale L. cv. Puma at cold-hardening temperatures with respect to leaf morphology, anatomy, and biochemistry, and with particular emphasis on their relationship to photosynthetic acclimation. Low temperature-induced structural and functional alterations at the level of ribulose bisphosphate carboxylase-oxygenase and the chloroplast thylakoid membrane are described and related to overall photosynthetic efficiency and capacity for CO₂ utilization. Growth and development at cold-hardening temperatures appear to result in changes in protein conformation and membrane organization, but not in basic composition. It is proposed that developmental temperature imparts a significant effect on the assembly of these multimeric, photosynthetic components, which leads to distinct structural and functional changes.     

The emergence of rabbit haemorrhagic disease virus: will a non-pathogenic strain protect the UK?

Rabbit haemorrhagic disease virus emerged in China in 1984, and has killed hundreds of millions of wild rabbits in Australia and Europe. In the UK there appears to be an endemic non-pathogenic strain, with high levels of seroprevalence being recorded, in the absence of associated mortality. Using a seasonal, age-structured model we examine the hypothesis that differences in rabbit population demography differentially affect the basic reproductive rates (R(0)) of the pathogenic and non-pathogenic strains, leading to each dominating in some populations and not others. The strain with the higher R(0) excluded the other, with the dynamics depending upon the ratio of the two R(0) values. When the non-pathogenic strain dominated, the pathogenic strain caused only transient mortality, although this could be significant when the two R(0) values were similar. When the pathogenic strain dominated, repeated epidemics led to host eradication. Seroprevalence data suggest that the non-pathogenic strain may be protecting some, but not all UK populations, with half being 'at risk' from invasion by the pathogenic strain and a fifth prone to significant transient mortality. We identify key questions for empirical research to test this prediction.     

Molecular characterization of microsporidia indicates that wild mammals Harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.