Development of anabolic-androgenic steroids purity certified reference materials for anti-doping

Background

The need for certified reference materials (CRM) of anabolic-androgenic steroids reference materials was emphasized by the Beijing 2008 Olympic game as a tool to improve comparability, ensuring accuracy and traceability of analytical results for competing athletes. The China National Institute of Metrology (NIM) responded to the state request by providing seven anabolic-androgenic steroids (AAS) reference materials for Beijing Olympic anti-doping, GBW (E) 100086-GBW (E) 100092.

Experimental

This work describes the production of the series of AAS CRMs, according to ISO Guides 34 and 35 [1] and [2], which comprises the material processing, homogeneity and stability assessment, CRMs’ characterization including moisture content, trace metal content. The AASs’ purity values were assigned with collaborative study involved eight laboratories applying high resolution liquid chromatography-diode array detector (HPLC-DAD). Homogeneity of the AAS CRMs were determined by an in-house validated liquid chromatographic methodology. Potential degradation during storage was also investigated and a shelf-life based on this value was established.

Results

The certified values of CRMs were 99.76 ± 0.079%, 99.76 ± 0.25%, 99.63 ± 0.09%, 99.67 ± 0.11%, 98.82 ± 0.56%, 96.30 ± 0.39% and 99.71 ± 0.49% (purity ± expanded uncertainty with confidence level of 95%) for methyltestosterone, testosterone propionate, nandrolone, nandrolone 17-propionate, boldenone, trenbolone acetate and testosterone respectively.

Conclusions

The certified values for all the studied AAS reference materials are traceable to the international system of units (SI). The CRMs developed were applied by 32 laboratory including sports organizations and analytical laboratories during the 2008 Olympic game for anti-doping control.     

Morphological and Molecular Differences in Two Strains of <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.     

Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52

cis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to d(?)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn²⁺-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an ¹⁸O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (β/α)₈ TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/β hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.     

5-酮基-D-葡萄糖酸发酵生产的工艺条件优化

葡萄糖酸氧化杆菌可将葡萄糖转化为5-酮基-D-葡萄糖酸(5-KGA),而5-KGA是重要食品添加剂L(+)-酒石酸的合成前体。为提高5-KGA产量及其对葡萄糖的转化率,对5-KGA发酵生产的工艺条件进行优化。在摇瓶水平最适的培养基和培养条件下,5-KGA最高产量为19.7 g/L,较优化前提高43.8%。在5 L发酵罐上控制恒定pH值5.5、溶氧浓度15%条件下,5-KGA产量达到46.0 g/L,较摇瓶最高产量提高1.3倍,应用葡萄糖流加工艺,5-KGA最高产量达到75.5 g/L,转化率超过70%,与已见报道的最高水平相比提高了32.0%,为实现微生物发酵生产5-KGA、进而合成L(+)-酒石酸的工业化提供了切实有效的途径。     

Probiotic Lactobacillus rhamnosus GG Enhanced Th1 Cellular Immunity but Did Not Affect Antibody Responses in a Human Gut Microbiota Transplanted Neonatal Gnotobiotic Pig Model

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 10⁹ colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 10⁶ CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.