Performance of survivin mRNA as a biomarker for bladder cancer in the prospective study UroScreen

Background

Urinary biomarkers have the potential to improve the early detection of bladder cancer. Most of the various known markers, however, have only been evaluated in studies with cross-sectional design. For proper validation a longitudinal design would be preferable. We used the prospective study UroScreen to evaluate survivin, a potential biomarker that has multiple functions in carcinogenesis.

Methods/Results

Survivin was analyzed in 5,716 urine samples from 1,540 chemical workers previously exposed to aromatic amines. The workers participated in a surveillance program with yearly examinations between 2003 and 2010. RNA was extracted from urinary cells and survivin was determined by Real-Time PCR. During the study, 19 bladder tumors were detected. Multivariate generalized estimation equation (GEE) models showed that β-actin, representing RNA yield and quality, had the strongest influence on survivin positivity. Inflammation, hematuria and smoking did not confound the results. Survivin had a sensitivity of 21.1% for all and 36.4% for high-grade tumors. Specificity was 97.5%, the positive predictive value (PPV) 9.5%, and the negative predictive value (NPV) 99.0%.

Conclusions

In this prospective and so far largest study on survivin, the marker showed a good NPV and specificity but a low PPV and sensitivity. This was partly due to the low number of cases, which limits the validity of the results. Compliance, urine quality, problems with the assay, and mRNA stability influenced the performance of survivin. However, most issues could be addressed with a more reliable assay in the future. One important finding is that survivin was not influenced by confounders like inflammation and exhibited a relatively low number of false-positives. Therefore, despite the low sensitivity, survivin may still be considered as a component of a multimarker panel.     

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 μg/ml) were found 3–4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS–PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sediment and Water Nutrient Characteristics in Patches of Submerged Macrophytes in Running Waters

(1) The relative importance of sediments and water as nutrient sources for submerged macrophytes in running waters is poorly understood. Here we present water and sediment nutrient characteristics within macrophyte patches in Bavarian rivers. (2) No significant differences between early (June/July) and late summer (August/September) sediment nutrient characteristics could be detected within macrophyte patches. Therefore, a single sediment sample per macrophyte patch was considered to be sufficient for characterising nutrient concentrations during the main growing season in running waters. (3) Sediment TP (total phosphorus) is not a useful parameter for predicting trophic status in running waters. Sediment porewater SRP (soluble reactive phosphorus) concentration is not correlated to water body SRP or TP concentration; nor is it correlated with sediment TP content. Potamogeton coloratus, a oligotrophic species, is associated with low overlying and porewater SRP concentrations but high sediment TP content. Eutrophic species, such as Potamogeton pectinatus, are associated with low sediment TP. (4) It is hypothesized that Chara hispida primarily takes up sediment ammonia for nitrogen nutrition. (5) Nutrient characteristics of the water body and the sediment of eight macrophyte species in Bavarian rivers are described.     

The dihydrolipoamide dehydrogenase from the crenarchaeon Acidianus ambivalens

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.     

Highly potent and selective alphaVbeta3-receptor antagonists: solid-phase synthesis and SAR of 1-substituted 4-amino-1H-pyrimidin-2-ones

Solid-phase synthesis and SAR of alpha(V)beta(3)-receptor antagonists based on a N(1)-substituted 4-amino-1H-pyrimidin-2-one scaffold are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta(3) in the nano- to subnanomolar range and high selectivity versus related integrins like alpha(IIb)beta(3). For selected examples efficacy in functional cellular assays was demonstrated.     

Acidianus ambivalens type-II NADH dehydrogenase: genetic characterisation and identification of the flavin moiety as FMN

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely (31)P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8alpha-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.     

DNA vaccines for allergy treatment

In the past 10 years, a great number of studies have demonstrated that injection of plasmid DNA coding for certain genes results in the induction of humoral and cellular immune responses against the respective gene product. This vaccination approach covers a broad range of possible applications, including the induction of protective immunity against viral, bacterial, and parasitic infections, and it opens new perspectives for treatment of cancer. Surprisingly, DNA immunization also turned out as a promising novel type of immunotherapy against allergy. In this paper, we describe the construction of DNA vaccines for application in allergy models. Beyond, we offer a palette of recently developed modulations to optimize DNA vaccines for allergy treatment by increasing their immunogenicity and minimizing their anaphylactic potential.     

A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer

Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret™, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E₂ production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1β. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1β either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1β produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1β synthesis.     

Ovulation Prevalence in Women with Spontaneous Normal-Length Menstrual Cycles – A Population-Based Cohort from HUNT3, Norway

Background

Ovulatory menstrual cycles are essential for women’s fertility and needed to prevent bone loss. There is a medical/cultural expectation that clinically normal menstrual cycles are inevitably ovulatory. Currently within the general population it is unknown the proportion of regular, normal-length menstrual cycles that are ovulatory. Thus, the objective of this study was to determine the population point prevalence of ovulation in premenopausal, normally menstruating women. The null hypothesis was that such cycles are ovulatory.

Methods

This is a single-cycle, cross-sectional, population-based study—a sub-study of the HUNT3 health study in the semi-rural county (Nord Trøndelag) in mid-Norway. Participants included >3,700 spontaneously (no hormonal contraception) menstruating women, primarily Caucasian, ages 20–49.9 from that county. Participation rate was 51.9%. All reported the date previous flow started. A single, random serum progesterone level was considered ovulatory if ≥9.54 nmol/L on cycle days 14 to -3 days before usual cycle length (CL).

Results

Ovulation was assessed in 3,168 women mean age 41.7 (interquartile range, [IQR] 36.8 to 45.5), cycle length 28 days (d) (IQR 28 to 28) and body mass index (BMI) 26.3 kg/m2 (95% CI 26.1 to 26.4). Parity was 95.6%, 30% smoked, 61.3% exercised regularly and 18% were obese. 1,545 women with a serum progesterone level on cycle days 14 to -3 were presumed to be in the luteal phase. Of these, 63.3% of women had an ovulatory cycle (n = 978) and 37% (n = 567) were anovulatory. Women with/ without ovulation did not differ in age, BMI, cycle day, menarche age, cigarette use, physical activity, % obesity or self-reported health. There were minimal differences in parity (96.7% vs. 94.5%, P = 0.04) and major differences in progesterone level (24.5 vs. 3.8 nmol/L, P = 0.001).

Conclusion

Anovulation in a random population occurs in over a third of clinically normal menstrual cycles.