Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.     

Lysosomotropic drugs inhibit maturation of transforming growth factor-beta

Transforming growth factor-beta (TGFbeta) is synthesized as a precursor protein, pro-TGFbeta, that must be cleaved by a furin-like proteinase before it becomes biologically active. We hypothesized that alkalinization of the trans-Golgi network (TGN)/endosome system may suppress pro-TGFbeta processing and decrease TGFbeta secretion. This hypothesis was tested in human A549 alveolar epithelial and T98G glioblastoma cell lines and in C57BL/6 mice. Inhibition of furin-like activity with decanoyl-RVKR chloromethylketone suppressed pro-TGFbeta processing, thereby significantly reducing the levels of secreted TGFbeta. Brefeldin A, bafilomycin A1, ammonium chloride, and monensin also prevented pro-TGFbeta processing. The alkalinizing lysosomotropic drugs chloroquine, hydroxychloroquine, amodiaquine, and azithromycin had a similar effect on the overall production of mature bioactive TGFbeta. Reduced levels of secreted TGFbeta were also associated with a decrease in Smad2 signaling. Mice treated with chloroquine showed a decrease in bronchoalveolar lavage fluid TGFbeta. We conclude that alkalinizing lysosomotropic drugs inhibit pro-TGFbeta processing.     

Microbial dynamics in glacier forefield soils show succession is not just skin deep

All over the world, glaciers are receding. One key consequence of glacier area loss is the creation of new terrestrial habitats. This presents an experimental opportunity to study both community formation and the implications of glacier loss for terrestrial ecosystems. In this issue of Molecular Ecology, Rime et al. ( 2015 ) describe how microbial communities are structured according to soil depth and development in the forefield of Damma glacier in Switzerland. The study provides insights into the contrasting structures of microbial communities at different stages of soil development. An important strength of the study is the integration of soil depth into the paradigm of primary succession, a feature which has rarely been considered by other studies. These findings underscore the importance of studying the interactions between microbial communities and glaciers at a time when Earth's glacial systems are experiencing profound change.     

Mitochondrial Reshaping Accompanies Neural Differentiation in the Developing Spinal Cord

Mitochondria, long known as the cell powerhouses, also regulate redox signaling and arbitrate cell survival. The organelles are now appreciated to exert additional critical roles in cell state transition from a pluripotent to a differentiated state through balancing glycolytic and respiratory metabolism. These metabolic adaptations were recently shown to be concomitant with mitochondrial morphology changes and are thus possibly regulated by contingencies of mitochondrial dynamics. In this context, we examined, for the first time, mitochondrial network plasticity during the transition from proliferating neural progenitors to post-mitotic differentiating neurons. We found that mitochondria underwent morphological reshaping in the developing neural tube of chick and mouse embryos. In the proliferating population, mitochondria in the mitotic cells lying at the apical side were very small and round, while they appeared thick and short in interphase cells. In differentiating neurons, mitochondria were reorganized into a thin, dense network. This reshaping of the mitochondrial network was not specific of a subtype of progenitors or neurons, suggesting that this is a general event accompanying neurogenesis in the spinal cord. Our data shed new light on the various changes occurring in the mitochondrial network during neurogenesis and suggest that mitochondrial dynamics could play a role in the neurogenic process.     

Blood biomarker discovery in drug-free schizophrenia: the contribution of proteomics and multiplex immunoassays

Introduction: Recent evidence supports an association between systemic abnormalities and the pathology of psychotic disorders which has led to the search for peripheral blood-based biomarkers.

Areas covered: Here, we summarize blood biomarker findings in schizophrenia from the literature identified by two methods currently driving biomarker discovery in the human proteome; mass spectrometry and multiplex immunoassay. From a total of 14 studies in the serum or plasma of drug-free schizophrenia patients; 47 proteins were found to be significantly altered twice or more, in the same direction. Pathway analysis was performed on these proteins, and the resulting pathways discussed in relation to schizophrenia pathology. Future directions are also discussed, with particular emphasis on the potential for high-throughput validation techniques such as data-independent analysis for confirmation of biomarker candidates.

Expert commentary: We present promising findings that point to a convergence of pathophysiological mechanisms in schizophrenia that involve the acute-phase response, glucocorticoid receptor signalling, coagulation, and lipid and glucose metabolism.     

The Cholinergic Anti-Inflammatory Pathway Delays TLR-Induced Skin Allograft Rejection in Mice: Cholinergic Pathway Modulates Alloreactivity

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.