Heterozygous mis-sense mutations in Prkcb as a critical determinant of anti-polysaccharide antibody formation

To identify rate-limiting steps in T cell-independent type 2 (TI-2) antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating ENU-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single amino acid substitutions within the kinase domain of Protein Kinase C Beta (PKCβ). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCβI whereas the amount of alternatively spliced PKCβII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to BCR-stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels

A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle

Abstract Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 µg kg-1. Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 µg kg-1, respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg-1 body weight per day for swine samples and 18 pg kg-1 body weight per day for turkey samples

Polyubiquitin binding to ABIN1 is required to prevent autoimmunity

The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor kappa B (NF-kappa B) essential modulator (NEMO), a component of the inhibitor of NF-kappa B (I kappa B) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88(-/-) mice, demonstrating that toll-like receptor (TLR)-MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-alpha/beta, c-Jun N-terminal kinases, and p38 alpha mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR-MyD88 pathways and prevent autoimmunity

Infusion of IL-10–expressing cells protects against renal ischemia through induction of lipocalin-2

Ischemia/reperfusion injury is a leading cause of acute renal failure triggering an inflammatory response associated with infiltrating macrophages, which determine disease outcome. To repair the inflammation we designed a procedure whereby macrophages that overexpress the anti-inflammatory agent interleukin (IL)-10 were adoptively transferred. These bone marrow–derived macrophages were able to increase their intracellular iron pool that, in turn, augmented the expression of lipocalin-2 and its receptors. Infusion of these macrophages into rats after 1h of reperfusion resulted in localization of the cells to injured kidney tissue, caused increases in regenerative markers, and a notable reduction in both blood urea nitrogen and creatinine. Furthermore, IL-10 therapy decreased the local inflammatory profile and upregulated the expression of pro-regenerative lipocalin-2 and its receptors. IL-10–mediated protection and subsequent renal repair were dependent on the presence of iron and lipocalin-2, since the administration of a neutralizing antibody for lipocalin-2 or administration of IL-10 macrophages pretreated with the iron chelating agent deferoxamine abrogated IL-10–mediated protective effects. Thus, adoptive transfer of IL-10 macrophages to ischemic kidneys blunts acute kidney injury. These effects are mediated through the action of intracellular iron to induce lipocalin-2

Follicular helper T cells are required for systemic autoimmunity

Production of high-affinity pathogenic autoantibodies appears to be central to the pathogenesis of lupus. Because normal high-affinity antibodies arise from germinal centers (GCs), aberrant selection of GC B cells, caused by either failure of negative selection or enhanced positive selection by follicular helper T (TFH) cells, is a plausible explanation for these autoantibodies. Mice homozygous for the san allele of Roquin, which encodes a RING-type ubiquitin ligase, develop GCs in the absence of foreign antigen, excessive TFH cell numbers, and features of lupus. We postulated a positive selection defect in GCs to account for autoantibodies. We first demonstrate that autoimmunity in Roquinsan/san (sanroque) mice is GC dependent: deletion of one allele of Bcl6 specifically reduces the number of GC cells, ameliorating pathology. We show that Roquinsan acts autonomously to cause accumulation of TFH cells. Introduction of a null allele of the signaling lymphocyte activation molecule family adaptor Sap into the sanroque background resulted in a substantial and selective reduction in sanroque TFH cells, and abrogated formation of GCs, autoantibody formation, and renal pathology. In contrast, adoptive transfer of sanroque TFH cells led to spontaneous GC formation. These findings identify TFH dysfunction within GCs and aberrant positive selection as a pathway to systemic autoimmunity

Flow topology of rare back flow events and critical points in turbulent channels and toroidal pipes

A study of the back flow events and critical points in the flow through a toroidal pipe at friction Reynolds number Re τ ≈ 650 is performed and compared with the results in a turbulent channel flow at Re τ ≈ 934. The statistics and topological properties of the back flow events are analysed and discussed. Conditionally-averaged flow fields in the vicinity of the back flow event are obtained, and the results for the torus show a similar streamwise wall-shear stress topology which varies considerably for the spanwise wall-shear stress when compared to the channel flow. The comparison between the toroidal pipe and channel flows also shows fewer back flow events and critical points in the torus. This cannot be solely attributed to differences in Reynolds number, but is a clear effect of the secondary flow present in the toroidal pipe. A possible mechanism is the effect of the secondary flow present in the torus, which convects momentum from the inner to the outer bend through the core of the pipe, and back from the outer to the inner bend through the pipe walls. In the region around the critical points, the skin-friction streamlines and vorticity lines exhibit similar flow characteristics with a node and saddle pair for both flows. These results indicate that back flow events and critical points are genuine features of wall-bounded turbulence, and are not artifacts of specific boundary or inflow conditions in simulations and/or measurement uncertainties in experiments.C. Chin, R. Vinuesa, R. Örlü, J. I. Cardesa, A. Noorani, P. Schlatter, and M. S. Chon

Novel lipochitin oligosaccharide structures produced by Rhizobium etli KIM5s

Microbial Biotechnolog

Genetic analysis of a pH-regulated operon from Rhizobium tropici CIAT899 involved in acid tolerance and nodulation competitiveness

Microbial Biotechnolog

Obtaining accurate mean velocity measurements in high Reynolds number turbulent boundary layers using Pitot tubes

This article reports on one component of a larger study on measurement of the zero-pressure-gradient turbulent flat plate boundary layer, in which a detailed investigation was conducted of the suite of corrections required for mean velocity measurements performed using Pitot tubes. In particular, the corrections for velocity shear across the tube and for blockage effects which occur when the tube is in close proximity to the wall were investigated using measurements from Pitot tubes of five different diameters, in two different facilities, and at five different Reynolds numbers ranging from Re_θ = 11 100 to 67 000. Only small differences were found amongst commonly used corrections for velocity shear, but improvements were found for existing near-wall proximity corrections. Corrections for the nonlinear averaging of the velocity fluctuations were also investigated, and the results compared to hot-wire data taken as part of the same measurement campaign. The streamwise turbulence-intensity correction was found to be of comparable magnitude to that of the shear correction, and found to bring the hot-wire and Pitot results into closer agreement when applied to the data, along with the other corrections discussed and refined here

Social network analysis as a tool to inform sustainable multi-sectoral management in complex marine socioecosystems

In complex marine socioecosystems, where multiple sectoral activities coexist, management can be especially complicated. This is the case of the coupled Guadalquivir estuary - Gulf of Cadiz (Ge-GoC) system. The Guadalquivir estuary (SW Spain) plays a central role in the whole Gulf of Cadiz since many fish species use these waters as a nursery area. Therefore, not only fisheries management is important but also the environmental status of the estuarine waters. The latter will have an effect on the adult populations via recruitment. Human activities that directly or indirectly influence the estuary include fishing, agriculture, shipping, saltworks, tourism or aquaculture among others. Conflict among them are common. In this context, we have used social network analyses as a first step to create an analysis and decision-support framework to achieve balanced socio-ecological ecosystem based management (EBM) to the Ge-GOC