Spore and micro-particle capture on an immunosensor surface in an ultrasound standing wave system

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator. Immunocapture in such a resonator has been characterised here for both spores and 1 microm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was x 70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10(4) particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.     

Activin receptor-like kinase 2 and Smad6 regulate epithelial-mesenchymal transformation during cardiac valve formation

Epithelial-mesenchymal transformation (EMT) occurs during both development and tumorigenesis. Transforming growth factor beta (TGFbeta) ligands signal EMT in the atrioventricular (AV) cushion of the developing heart, a critical step in valve formation. TGFbeta signals through a complex of type I and type II receptors. Several type I receptors exist although activin receptor-like kinase (ALK) 5 mediates the majority of TGFbeta signaling. Here, we demonstrate that ALK2 is sufficient to induce EMT in the heart. Both ALK2 and ALK5 are expressed throughout the heart with ALK2 expressed abundantly in endocardial cells of the outflow tract (OFT), ventricle, and AV cushion. Misexpression of constitutively active (ca) ALK2 in non-transforming ventricular endocardial cells induced EMT, while caALK5 did not, thus demonstrating that ALK2 activity alone is sufficient to stimulate EMT. Smad6, an inhibitor of Smad signaling downstream of ALK2, but not ALK5, inhibited EMT in AV cushion endocardial cells. These data suggest that ALK2 activation may stimulate EMT in the AV cushion and that Smad6 may act downstream of ALK2 to negatively regulate EMT.     

Comparison of microsatellites versus single-nucleotide polymorphisms in a genome linkage screen for prostate cancer-susceptibility Loci

Prostate cancer is one of the most common cancers among men and has long been recognized to occur in familial clusters. Brothers and sons of affected men have a 2-3-fold increased risk of developing prostate cancer. However, identification of genetic susceptibility loci for prostate cancer has been extremely difficult. Although the suggestion of linkage has been reported for many chromosomes, the most promising regions have been difficult to replicate. In this study, we compare genome linkage scans using microsatellites with those using single-nucleotide polymorphisms (SNPs), performed in 467 men with prostate cancer from 167 families. For the microsatellites, the ABI Prism Linkage Mapping Set version 2, with 402 microsatellite markers, was used, and, for the SNPs, the Early Access Affymetrix Mapping 10K array was used. Our results show that the presence of linkage disequilibrium (LD) among SNPs can lead to inflated LOD scores, and this seems to be an artifact due to the assumption of linkage equilibrium that is required by the current genetic-linkage software. After excluding SNPs with high LD, we found a number of new LOD-score peaks with values of at least 2.0 that were not found by the microsatellite markers: chromosome 8, with a maximum model-free LOD score of 2.2; chromosome 2, with a LOD score of 2.1; chromosome 6, with a LOD score of 4.2; and chromosome 12, with a LOD score of 3.9. The LOD scores for chromosomes 6 and 12 are difficult to interpret, because they occurred only at the extreme ends of the chromosomes. The greatest gain provided by the SNP markers was a large increase in the linkage information content, with an average information content of 61% for the SNPs, versus an average of 41% for the microsatellite markers. The strengths and weaknesses of microsatellite versus SNP markers are illustrated by the results of our genome linkage scans.     

The CARMA3-Bcl10-MALT1 Signalosome Promotes Angiotensin II-dependent Vascular Inflammation and Atherogenesis

The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-κB in lymphocytes, thereby representing a cornerstone of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-κB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-κB in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known.     

Amoebal host range, host-free survival and disinfection susceptibility of environmental Chlamydiae as compared to Chlamydia trachomatis

The term 'Chlamydia-like organisms' encompasses obligate intracellular bacterial species phylogenetically close to Chlamydiaceae. Most are associated with free-living amoebae, and several could be responsible for respiratory tract infections and abortion in human and animals. Despite increasing concern about their pathogenic role, the prevalence, biodiversity and ecology of Chlamydia-related bacteria still remain largely unknown. In this study, six members of the Chlamydiales were tested, including Parachlamydia acanthamoebae (two different strains), Protochlamydia naegleriophila, Waddlia chondrophila, Criblamydia sequanensis and Chlamydia trachomatis as a reference. Intracellular growth was tested in 11 different Acanthamoeba strains, demonstrating significant differences in host susceptibilities to infection depending on strains investigated. Survival of host-free bacteria in suspension or dried onto surfaces was also explored, demonstrating that Chlamydia-like organisms present better survival capacity than C. trachomatis. Longer survival times were observed for bacteria suspended in rich culture medium, with survivors being detected after 10 weeks incubation. We also tested susceptibility of host-free Chlamydia-like organisms to several disinfection treatments. Each chemical biocide tested reduced viability of host-free Chlamydia by more than 4 logs. Conversely, all Chlamydia-like organisms tested resisted exposure at 55 °C for 10 min, while C. trachomatis was completely inactivated.     

The species–area relationship in centipedes (Myriapoda: Chilopoda): a comparison between Mediterranean island groups

The present study article examines the shapes of centipede species–area relationships (SARs) in the Mediterranean islands, compares the results of the linear form of the power model between archipelagos, discusses biological significance of the power model parameters with other taxa on the Aegean archipelago, and tests for a significant small‐island effect (SIE). We used 11 models to test the SARs and we compared the quality‐of‐fit of all candidate models. The power function ranked first and Z‐values was in the range 0.106–0.334. We assessed the presence of SIEs by fitting both a continuous and discontinuous breakpoint regression model. The continuous breakpoint regression functions never performed much better than the closest discontinuous model as a predictor of centipede species richness. We suggest that the relatively low Z‐values in our data partly reflect better dispersal abilities in centipedes than in other soil invertebrate taxa. Longer periods of isolation and more recent island formation may explain the somewhat lower constant c in the western Mediterranean islands compared to the Aegean islands. Higher breakpoint values in the western Mediterranean may also be a result of larger distance to the mainland and longer separation times. Despite the differences in the geological history and the idiosyncratic features of the main island groups considered, the overall results are quite similar and this could be assigned to the ability of centipedes to disperse across isolation barriers. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 146–159.     

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.