Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.     

Dietary Benefits of Twilight Foraging by the Insectivorous Bat Hipposideros speoris1

Although bats are nocturnal, many species emerge from roosts to forage during twilight, despite a presumed high risk of predation at this time. Here, we describe twilight foraging by a maternity colony of Schneider's leafnosed bat (Hipposideros speoris) in the dry zone of Sri Lanka and determine the dietary benefits of such behavior. Bats usually began foraging during dusk, sometimes before sunset, and also foraged during twilight in the morning. Mean use of available twilight by four radio‐tagged bats was 75 percent. Twilight foraging made up, on average, 47 percent of the total foraging time of these bats (range = 25–96%), although twilight consisted of only 12 percent of the available time between sunset and sunrise the next morning. Eight species of potential predators (7 birds and 1 mammal) were observed within a 1 km radius of the colony, of which 5 species are predicted to regularly capture bats. Bats took a wide diversity of prey (11 insect orders, including at least 27 families, and spiders) that ranged in wing length from 2.0 to 54.0 mm. Major orders in the diet were Coleoptera, Lepidoptera, and Diptera. Prey of secondary importance included Hemiptera, Hymenoptera, Isoptera, and Neuroptera. Bats captured large numbers of insects that were only available or had marked peaks in abundance during twilight. These groups included small, swarming insects (especially flies) that have peaks in flight activity at dusk and dawn, large diurnal species (especially dragonflies) that have crepuscular activity, and winged termites that emerge in swarms at dusk. Access to these insects was a clear benefit of twilight foraging.     

Microbial carbonate crusts-a key to the environmental analysis of fossil spongiolites?

Summary Morphological and geochemical comparisons between modern cryptic microbialites from Lizard Island/Great Barrier Reef and fossil counterparts in the Upper Jurassic (Southern Germany, Dobrogea/Romania) and late Lower Cretaceous (Aptian/Albian from Cantabria/Spain) spongiolitic environments show that there are common factors controlling the crust formations mostly independent of light despite of diverging (paleo-) oceanographic positions as well as relationships of competitors. Factors such as increased alkalinity, oligotrophy, and reduced allochthonous deposition are of major importance. Thrombolitic microbialites are interpreted as biologically induced and therefore calcified in isotopic equilibrium with the surrounding sea water. Corresponding with shallowing upward cycles, microbial mats which produce stromatolitic peloidal crusts become more important. Different biomarkers are introduced for the first time extracted and analyzed from spongiolitic limestones of Lower Kimmeridgian age from Southern Germany.     

Approximate Bayesian Computation Untangles Signatures of Contemporary and Historical Hybridization between Two Endangered Species

Contemporary gene flow, when resumed after a period of isolation, can have crucial consequences for endangered species, as it can both increase the supply of adaptive alleles and erode local adaptation. Determining the history of gene flow and thus the importance of contemporary hybridization, however, is notoriously difficult. Here, we focus on two endangered plant species, Arabis nemorensis and A. sagittata, which hybridize naturally in a sympatric population located on the banks of the Rhine. Using reduced genome sequencing, we determined the phylogeography of the two taxa but report only a unique sympatric population. Molecular variation in chloroplast DNA indicated that A. sagittata is the principal receiver of gene flow. Applying classical D-statistics and its derivatives to whole-genome data of 35 accessions, we detect gene flow not only in the sympatric population but also among allopatric populations. Using an Approximate Bayesian computation approach, we identify the model that best describes the history of gene flow between these taxa. This model shows that low levels of gene flow have persisted long after speciation. Around 10 000 years ago, gene flow stopped and a period of complete isolation began. Eventually, a hotspot of contemporary hybridization was formed in the unique sympatric population. Occasional sympatry may have helped protect these lineages from extinction in spite of their extremely low diversity.     

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C⁺ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C⁺ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C⁺ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2C^wt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2C^del/del) and heterozygous (NKG2C^wt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C⁺ cells. We therefore analyzed the PUUV-mediated NKG2C⁺ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C⁺ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C⁺ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2C^del and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2C^wt/del allele was significantly overrepresented, compared to the NKG2C^wt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2C^wt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2C^wt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C⁺ NK cell response, as reflected by NKG2C^wt/del variant, may be associated with a higher risk for a severe hantavirus infections.     

Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease

The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients’ sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA^H) and speckled ANA (ANA^S) staining pattern, and also steroid-responsive meningitis-arteritis (SRMA) are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAMTOR3, DDIT4L and PPP3CA) located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.     

The serotoninergic receptors of human dendritic cells: identification and coupling to cytokine release

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is stored at peripheral sites in mast cells and released from this peripheral source upon IgE cross-linking. In this study, we investigated the expression of serotoninergic receptors (5-HTR), the signaling pathway, and biological activity of 5-HT on human dendritic cells (DC), showing that immature and mature DC expressed mRNA for different serotoninergic receptors. Thereby, the mRNA of 5-HTR(1B), 5-HTR(1E), 5-HTR(2A), 5-HTR(2B), one splicing variant of the 5-HTR(3), 5-HTR(4), and 5-HTR(7) receptors were detected. Immature DC preferentially expressed mRNA for the heptahelical 5-HTR(1B), 5-HTR(1E), and 5-HTR(2B) receptors, while mature DC mostly expressed 5-HTR(4) and 5-HTR(7). The mRNA expression level of the ligand-gated cation channel 5-HTR(3) and the heptahelical 5-HTR(2A) did not significantly change during maturation. Isotype-selective receptor agonists allowed us to show that 5-HT stimulated 5-HTR(3)-dependent Ca(2+) influx in immature and mature DC. Moreover, we revealed that 5-HTR(1) and 5-HTR(2) receptor stimulation induced intracellular Ca(2+) mobilization via G(i/o) proteins in immature, but not mature, DC. Activation of 5-HTR(4) and 5-HTR(7) induced cAMP elevation in mature DC. Functional studies indicated that activation of 5-HTR(4) and 5-HTR(7) enhanced the release of the cytokines IL-1beta and IL-8, while reducing the secretion of IL-12 and TNF-alpha in mature DC. In summary, our study shows that 5-HT stimulated, in a maturation-dependent manner, different signaling pathways in DC. These data point to a role for 5-HT in regulating the immune response at peripheral sites.     

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH(i)), we suggest that F-actin disruption affected Na(+)/H(+) exchanger activity. In line with this, pH(i) recovery after an ammonium prepulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na(+) under hypertonic conditions was not diminished but, rather, enhanced by F-actin disruption, presumably due to inhibited Na(+)-K(+)-ATPase activity and stimulated Na(+) channel activity. The elevation of cytosolic Ca(2+) in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.     

Catalytically Increased Prebiotic Peptide Formation: Ditryptophan,Dilysine, and Diserine

“Mutual” amino acid catalysis of glycine on the formation of ditryptophan, dilysine, and diserine in the prebiotically relevant Salt-Induced Peptide Formation (SIPF) Reaction was investigated varying the starting concentration and chirality of the educt amino acid, and analyzing the increase of yield resulting from this catalytic effect. Our results show the possibility of an amplified diverse pool of peptides being available for chemical evolution of larger peptides and proteins using also these more complicated amino acids for the evolution of more complex functions in future biochemical cycles and thus for the emergence of life. Catalytic effects are especially high in the case of serine, the most basic amino acid of the three, but are also significant for the other two examples investigated in the present work. Besides that, especially for serine, but also in the case of tryptophan, differences in catalytic yield increase according to the chiral form of the amino acid used could be observed.