DISPERSAL AND POPULATION-GENETIC STRUCTURE OF THE COOPERATIVE SPIDER,AGELENA CONSOCIATA,IN WEST AFRICAN RAINFOREST

Dispersal experiments and gel electrophoresis of allozyme polymorphisms were used to investigate the selective mode underlying cooperative behavior in the rainforest spider, Agelena consociata. Previous work has indicated that individual selection alone does not explain the cooperative and even altruistic behavior noted for this African species, which exists in groups of up to hundreds of adults. We found no evidence for active dispersal by reproductives or any age class of this spider. Nest fragmentation by falling tree limbs and storms is indicated as the cause of new nest formation within local areas, while passive dispersal by vertebrate carriers that either have some association with the nests (bats) or move through them is indicated as the probable mode of longer-distance dispersal. The population-genetic structure observed for A. consociata supports the data obtained on dispersal. Wright's F_ST statistic and G tests for genetic heterogeneity indicate that the populations are subdivided into genetically heterogeneous colonies. Comparisons utilizing Nei's genetic distance show colonies separated by as few as 30 m to be as genetically distinct as are colonies separated by many kilometers. There is also a marked scarcity of heterozygotes, and individuals within nests and associated colonies are genetically related about as much as are full siblings. The results of these analyses indicate that kin selection or some type of family-group selection may have been important in the evolution of cooperative behavior in the species.     

Glycerol supplementation enhances L. reuteri's protective effect against S. Typhimurium colonization in a 3-D model of colonic epithelium

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.     

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.     

Systems Biology Analysis Merging Phenotype,Metabolomic and Genomic Data Identifies Non-SMC Condensin I Complex,Subunit G (NCAPG) and Cellular Maintenance Processes as Major Contributors to Genetic Variability in Bovine Feed Efficiency

Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as observed in our study.     

Kinetic alterations due to a missense mutation in the Na,K-ATPase alpha2 subunit cause familial hemiplegic migraine type 2

A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase alpha2 subunit, have been identified in familial hemiplegic migraine with aura. Loss of function and haploinsufficiency have been the suggested mechanisms in mutants for which functional analysis has been reported. This paper describes a kinetic analysis of mutant T345A, recently identified in a detailed genetic analysis of a large Finnish family (Kaunisto, M. A., Harno, H., Vanmolkot, K. R., Gargus, J. J., Sun, G., Hamalainen, E., Liukkonen, E., Kallela, M., van den Maagdenberg, A. M., Frants, R. R., Farkkila, M., Palotie, A., and Wessman, M. (2004) Neurogenetics 5, 141-146). Introducing T345A into the conserved rat alpha2 enzyme does not alter cell growth or catalytic turnover but causes a substantial decrease in apparent K+ affinity (2-fold increase in K0.5(K+)). In view of the location of Thr-345 in the cytoplasmic stalk domain adjacent to transmembrane segment 4, the 2-fold increase in K0.5(K+) is probably due to T345A replacement altering K+ occlusion/deocclusion. Faster K+ deocclusion of the mutant via the E2(K) + ATP --> E1.ATP + K+ partial reaction is evidenced in (i) a marked increase (300%) in K+ stimulation of Na-ATPase at micromolar ATP, (ii) a 4-fold decrease in KATP, and (iii) only a modest increase (approximately 3-fold) in I50 for vanadate, which was used as a probe of the steady state E1/E2 conformational equilibrium. We suggest that the decreased apparent K+ affinity is the basis for a reduced rate of extracellular K+ removal, which delays the recovery phase of nerve impulse transmission in the central nervous system and, thereby, the clinical picture of migraine with aura. This is the first demonstration of a mutation that leads to a disease associated with a kinetically altered but fully functional Na,K-ATPase, refining the molecular mechanism of pathogenesis in familial hemiplegic migraine.     

Homeostasis of peripheral FoxP3+ CD4+ regulatory T cells in patients with early and late stage breast cancer

FoxP3 ⁺ CD4 ⁺ regulatory T cells (Tregs) are important mediators of peripheral immune tolerance, acting via multiple mechanisms to suppress cellular immunity including antitumor responses. Although therapeutic strategies have been proposed to deplete Tregs in patients with breast cancer and other malignancies, dynamic changes in the Treg compartment as a function of stage and treatment of breast cancer remain poorly understood. Here, we evaluated peripheral blood CD4⁺ T cells and FoxP3⁺ CD4⁺ T cells from 45 patients with early or late stage breast cancer and compared percentages, absolute counts, and Treg function to those from healthy volunteers (HV) of comparable age. Patients having completed adjuvant chemotherapy and patients with metastatic cancer exhibited significantly lower absolute CD4 counts and significantly higher percentages of FoxP3⁺ CD4⁺ T cells. In contrast, the absolute counts of circulating FoxP3⁺ CD4⁺ T cells did not differ significantly among early stage patients, late stage patients, or HV. Functionally, FoxP3⁺ CD4⁺ T cells from all donor groups similarly expressed CTLA-4 and failed to secrete IFN-γ in response to stimulation. Thus, although Tregs comprise an increased percentage of circulating CD4⁺ T cells in patients with metastatic breast cancer and patients in remission after completing the adjuvant chemotherapy, the systemic Treg pool, as measured by absolute counts, appears relatively constant regardless of disease stage or treatment status. Total CD4⁺ T cell counts are not constant, however, suggesting that homeostatic mechanisms, or susceptibility to cytotoxic or malignant insults, fundamentally differ for regulatory and non-regulatory CD4⁺ T cells.     

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction.     

Biology and host specificity of Rhinusa pilosa,a recommended biological control agent of Linaria vulgaris

Linaria vulgaris Mill. (Plantaginaceae), common or yellow toadflax, is a Eurasian short-lived perennial forb invasive throughout temperate North America. Rhinusa pilosa (Gyllenhal) (Coleoptera, Curculionidae) is a univoltine shoot-galling weevil found exclusively on L. vulgaris in Europe. Under no-choice test conditions, 13 non-native Linaria species exposed to R. pilosa were accepted for oviposition and most were found to be suitable, to varying degrees, for gall and larval development. Adult feeding and survival was minimal on native North American species in the plant tribe Antirrhineae which includes the target plant. In no-choice tests with 63 native North American species and 24 other non-target species outside Linaria, oviposition was limited to four native North American species. Only three larvae developed to the adult stage on Sairocarpus virga (A. Gray) D.A. Sutton, with no negative impact on plant growth. Risks to native flora from the release of R. pilosa are therefore expected to be minimal. The Technical Advisory Group for the Biological Control of Weeds (TAG—BCW) has recommended release of R. pilosa in September 2013.     

Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

Background

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.

Methods

Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.

Results

Correlation of IFN- γ⁺ spot-forming cells in EliSpot-IGRA and FluoroSpot were R² = 0.67 for ESAT-6 and R² = 0.73 for CFP-10. The number of IL-2^- IFN- γ⁺ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ⁺ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.

Conclusions

Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.