Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

Full-length IL-33 is a member of the IL-1 family of cytokines, which can act in an autocrine or paracrine manner by binding to the IL-33R on several different target cell types. In addition, IL-33 can act in an intracrine fashion by translocating to the nucleus, where it binds to the chromatin and modulates gene expression. In this article, we report that full-length IL-33, but not mature IL-33, interacts with the transcription factor NF-κB. This interaction occurs between the N-terminal part of IL-33 from aa 66–109 and the N-terminal Rel homology domain of NF-κB p65. Coimmunoprecipitation experiments in cells overexpressing IL-33 or endogenously expressing IL-33 revealed rhIL-1β–stimulated association between IL-33 and p65, whereas binding to the p50 subunit was constitutive. The biological consequence of IL-33/NF-κB complex formation was reduction in NF-κB p65 binding to its cognate DNA and impairment of p65-triggered transactivation. Overexpression of IL-33 resulted in a reduction and delay in the rhIL-1β–stimulated expression of endogenous NF-κB target genes such as IκBα, TNF-α, and C-REL. We suggest that nuclear IL-33 sequesters nuclear NF-κB and reduces NF-κB–triggered gene expression to dampen proinflammatory signaling.

BackgroundIdentifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals.ResultsHerein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%).ConclusionWe strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

Population and locus-specific reduction of variability of polymorphic loci could be an indication of positive selection at a linked site (selective sweep) and therefore point toward genes that have been involved in recent adaptations. Analysis of microsatellite variability offers a way to identify such regions and to ask whether they occur more often than expected by chance. We studied four populations of the house mouse (Mus musculus) to assess the frequency of such signatures of selective sweeps under natural conditions. Three samples represent the subspecies Mus m. dometicus [corrected] and came from Germany, France, and Cameroon. One sample came from Kazakhstan and constitutes a population of the subspecies Mus m. [corrected] musculus. Mitochondrial D-loop sequences from all animals confirm their respective assignments. Approximately 200 microsatellite loci were typed for up to 60 unrelated individuals from each population and evaluated for signs of selective sweeps on the basis of Schlötterer's ln RV and ln RH statistics. Our data suggest that there are slightly more signs of selective sweeps than would have been expected by chance alone in each of the populations and also highlights some of the statistical challenges faced in genome scans for detecting selection. Single-nucleotide polymorphism typing of one sweep signature in the M. m. domesticus populations around the beta-defensin 6 locus confirms a lowered nucleotide diversity in this region and limits the potential sweep region to about 20 kb. However, no amino acid exchange has occurred in the coding region when compared to M. m. musculus. If this sweep signature is due to a recent adaptation, it is expected that a regulatory change would have caused it. Our data provide a framework for conducting a systematic whole genome scan for signatures of selective sweeps in the mouse genome.

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed-in contrast to viable parasites-that apoptotic-like parasites enter an LC3 C , autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4 C T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferationreducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

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We have evaluated a pooling approach that can reduce the number of polymerase chain reactions in a screen for selective sweeps by more than an order of magnitude. We show that the complex peak pattern that results from pooling of all samples from a given population is a faithful reflection of the composite pattern of the individual alleles, although with an under‐representation of the larger alleles. Candidate loci for selective sweeps can be identified by visual inspection of the pool patterns. We have also implemented a software tool, which can find suitable microsatellite loci in the vicinity of annotated genes.

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