Assessment of composition and anisotropic elastic properties of secondary osteon lamellae

Measurement of the elastic properties of single osteon lamellae is still one of the most demanding tasks in bone mechanics to be solved. By means of site-matched Raman microspectroscopy, acoustic microscopy and nanoindentation the structure, chemical composition and anisotropic elasticity of individual lamellae in secondary osteons were investigated. Acoustic impedance images (911-MHz) and two-dimensional Raman spectra were acquired in sections of human femoral bone. The samples were prepared with orientations at various observation angles theta relative to the femoral long axis. Nanoindentations provided local estimations of the elastic modulus and landmarks necessary for spatial fusion of the acoustic and spectral Raman images. Phosphate nu(1) (961 cm(-1)) and amide I (1665 cm(-1)) band images representing spatial distributions of mineral and collagen were fused with the acoustic images. Acoustic impedance was correlated with the indentation elastic modulus E(IT) (R(2)=0.61). Both parameters are sensitive to elastic tissue anisotropy. The lowest values were obtained in the direction perpendicular to the femoral long axis. Acoustic images exhibit a characteristic bimodal lamellar pattern of alternating high and low impedance values. Since this undulation was not associated with a variation of the phosphate nu(1)-band intensity in the Raman images, it was attributed to variations of the lamellar orientation. After threshold segmentation and conversion to elastic modulus the orientation and transverse isotropic elastic constants were derived for individual ensembles of apparent thin and thick lamellae. Our results suggest that this model represents the effective anisotropic properties of an asymmetric twisted plywood structure made of transverse isotropic fibrils. This is the first report that proves experimentally the ability of acoustic microscopy to map tissue elasticity in two dimensions with micrometer resolution. The combination with Raman microspectroscopy provides a unique way to study bone and mineral metabolism and the relation with mechanical function at the ultrastructural tissue level.     

Physiological diversity of Roseobacter clade bacteria co-occurring during a phytoplankton bloom in the North Sea

Organisms of the Roseobacter clade are an important component in marine ecosystems, partially due to their metabolic variety. Not much is known, however, about the physiological diversity of different roseobacters present within one habitat. By using serial dilution cultures with low-nutrient media seven roseobacter strains, co-occurring during a phytoplankton bloom in the southern North Sea, were obtained in this study. Physiological characterization exhibited distinct substrate spectra of the isolates. Although no isolate showed growth on algal osmolyte dimethylsulfoniopropionate (DMSP), feeding experiments revealed that all new strains converted [²H₆]DMSP into a variety of volatile compounds. Six strains mainly decomposed DMSP via the demethylation pathway, but four strains were also capable of cleaving DMSP to DMS and acrylate. It is hypothesized that the great physiological diversity of the roseobacters reflects their ability to inhabit different ecological niches and enables the organisms to cope differently with changing substrate supplies during phytoplankton blooms. Denaturing gradient gel electrophoresis and sequencing of excised bands resulted in detection of five additional roseobacters. Three of these sequences showed affiliation with three of the four major clusters of the Roseobacter clade, consisting predominantly of uncultured organisms (i.e. the Roseobacter clade-affiliated (RCA)), the NAC11-7 and the CHAB-I-5 clusters.     

Genome sequence of Phaeobacter caeruleus type strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564^T was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.     

Blinded sample size recalculation in multicentre trials with normally distributed outcome

The internal pilot study design enables to estimate nuisance parameters required for sample size calculation on the basis of data accumulated in an ongoing trial. By this, misspecifications made when determining the sample size in the planning phase can be corrected employing updated knowledge. According to regulatory guidelines, blindness of all personnel involved in the trial has to be preserved and the specified type I error rate has to be controlled when the internal pilot study design is applied. Especially in the late phase of drug development, most clinical studies are run in more than one centre. In these multicentre trials, one may have to deal with an unequal distribution of the patient numbers among the centres. Depending on the type of the analysis (weighted or unweighted), unequal centre sample sizes may lead to a substantial loss of power. Like the variance, the magnitude of imbalance is difficult to predict in the planning phase. We propose a blinded sample size recalculation procedure for the internal pilot study design in multicentre trials with normally distributed outcome and two balanced treatment groups that are analysed applying the weighted or the unweighted approach. The method addresses both uncertainty with respect to the variance of the endpoint and the extent of disparity of the centre sample sizes. The actual type I error rate as well as the expected power and sample size of the procedure is investigated in simulation studies. For the weighted analysis as well as for the unweighted analysis, the maximal type I error rate was not or only minimally exceeded. Furthermore, application of the proposed procedure led to an expected power that achieves the specified value in many cases and is throughout very close to it.     

Proteomics reveals potential biomarkers of seed vigor in sugarbeet

To unravel biomarkers of seed vigor, an important trait conditioning crop yield, a comparative proteomic study was conducted with sugarbeet seed samples of varying vigor as generated by an invigoration treatment called hydropriming and an aging treatment called controlled deterioration. Comparative proteomics revealed proteins exhibiting contrasting behavior between seed samples. Thus, 18 proteins were up-regulated during priming and down-regulated during aging and further displayed an up-regulation upon priming of the aged seeds, meaning that down-regulation of these spot volumes during aging was reversible upon subsequent priming. Also, 11 proteins exhibited the converse behavior characterized by a decrease and an increase of the spot volumes during priming and aging of the control seeds, respectively, and a decrease in the spot volumes upon priming of the aged seeds. The results underpinned the role in seed vigor of several metabolic pathways involved in lipid and starch mobilization, protein synthesis or the methyl cycle. They also corroborate previous studies suggesting that the glyoxylate enzyme isocitrate lyase, the capacity of protein synthesis and components of abscisic acid signaling pathways are likely contributors of seed vigor.     

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.     

QuickLIMS: facilitating the data management for DNA-microarray fabrication

SUMMARY: QuickLIMS is a Microsoft Access-based laboratory information and management system, capable of processing all information for microarray production. The program's operational flow is protocol-based, dynamically adapting to changes of the process. It interacts with the laboratory robot and with other database systems over the network, and it represents a complete solution for the management of the entire manufacturing process. AVAILABILITY AND SUPPLEMENTARY INFORMATION: http://www.dkfz-heidelberg.de/kompl_genome/Other/QuickLims/index.html