The male reproductive tract has been identified as a target tissue for vitamin D, and previous data suggest an association of 25-hydroxyvitamin D [25(OH)D] with testosterone levels in men. We therefore aimed to evaluate whether vitamin D supplementation influences testosterone levels in men. Healthy overweight men undergoing a weight reduction program who participated in a randomized controlled trial were analyzed for testosterone levels. The entire study included 200 nondiabetic subjects, of whom 165 participants (54 men) completed the trial. Participants received either 83 μg (3 332 IU) vitamin D daily for 1 year (n=31) or placebo (n=23). Initial 25(OH)D concentrations were in the deficiency range (<50 nmol/l) and testosterone values were at the lower end of the reference range (9.09–55.28 nmol/l for males aged 20–49 years) in both groups. Mean circulating 25(OH)D concentrations increased significantly by 53.5 nmol/l in the vitamin D group, but remained almost constant in the placebo group. Compared to baseline values, a significant increase in total testosterone levels (from 10.7±3.9 nmol/l to 13.4±4.7 nmol/l; p<0.001), bioactive testosterone (from 5.21±1.87 nmol/l to 6.25±2.01 nmol/l; p=0.001), and free testosterone levels (from 0.222±0.080 nmol/l to 0.267±0.087 nmol/l; p=0.001) were observed in the vitamin D supplemented group. By contrast, there was no significant change in any testosterone measure in the placebo group. Our results suggest that vitamin D supplementation might increase testosterone levels. Further randomized controlled trials are warranted to confirm this hypothesis.

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated.

The risk of transfusion-transmitted hepatitis E virus (HEV) infections by contaminated blood products remains unknown. In the present study, we evaluated and compared different nucleic acid amplification technique (NAT) methods for the detection of HEV in blood components. Minipools of a total of 16,125 individual blood donors were screened for the presence of HEV RNA using the highly sensitive RealStar HEV RT-PCR kit, revealing a minimum detection limit of 4.66 IU/ml. Thirteen donors were HEV RNA positive (0.08%), and of these donors, only three already showed reactive IgM antibody titers. The detected HEV strains all belonged to genotype 3 and were most closely related to German HEV strains from wild boars and pigs as well as from human hepatitis E cases. Furthermore, HEV RNA and HEV-specific IgM and IgG titers were determined in 136 blood donors with elevated alanine aminotransferase (ALT) levels and in 200 donors without pathological findings. HEV RNA was not detectable, but 8.08% (elevated ALT) and 0.5% (nonelevated ALT) of donors showed reactive HEV IgM titers. The overall seroprevalence rate of HEV IgG amounted to 5.94% (elevated ALT, 5.88%; nonelevated ALT, 6.0%). The clinical relevance of transfusion-associated hepatitis E infection still requires further investigation. However, in connection with raising concerns regarding blood safety, our NAT method provides a sensitive possibility for HEV testing.

The phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D. Meletzus, A. Bermpohl, J. Dreier, and R. Eichenlaub, 1993, J. Bacteriol. 175:2131–2136; J. Dreier, D. Meletzus, and R. Eichenlaub, 1997, Mol. Plant-Microbe Interact. 10:195–206). The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation. RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped. The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-β-1,4-glucanases of family A₁ cellulases. CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain. We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA^- gene-replacement mutant CMM-H4 with the wild-type celA gene.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype.

A significant proportion of cardiac surgical patients develop critical post-operative complications. We aimed to investigate the association of pre-operative 25-hydroxyvitamin D (25(OH)D) levels with major cardiac and cerebrovascular events (MACCE) in cardiac surgical patients. From January 2010 to August 2011, we consecutively measured circulating 25(OH)D in 4418 operated patients. Of the study cohort, 38.0% had deficient 25(OH)D values (100 nmol/L. The incidence of MACCE was 11.5%. In multivariable-adjusted logistic regression models, the odds ratio of MACCE at deficient, inadequate, and high 25(OH)D levels was 2.23 [95% confidence interval (CI): 1.31–3.79], 1.73 (95% CI: 1.01–2.96) and 2.34 (95% CI: 1.12–4.89), respectively, compared with 25(OH)D levels of 75–100 nmol/L. A U-shaped association with circulating 25(OH)D was also present for duration of mechanical ventilatory support and intensive care unit stay. Multivariable-adjusted 6- and 12-month mortality were higher in patients with deficient 25(OH)D levels compared with patients with 25(OH)D levels of 75–100 nmol/L. Deficient 25(OH)D levels are prevalent in cardiac surgical patients in Central Europe and are independently associated with the risk of MACCE. Further research should clarify the potential of vitamin D supplements in reducing cardiovascular risk in vitamin D-deficient patients and also the mechanisms leading to adverse effects on the cardiovascular system in the small group of patients with 25(OH)D levels >100 nmol/L. Trial registration information: Clinicaltrials.gov identifier number: NCT01552382.