ALBERT

Description: Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis -spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome.

Description: Target DNA enrichment combined with high-throughput sequencing technologies is a powerful approach to probing a large number of loci in genomes of interest. However, software algorithms that explicitly consider nucleotide sequence information of target loci in multiple reference species for optimizing design of target enrichment baits to be applicable across a wide range of species have not been developed. Here we present an algorithm that infers target DNA enrichment baits from multiple nucleotide sequence alignments. By applying clustering methods and the combinatorial 1-center sequence optimization to bait design, we are able to minimize the total number of baits required to efficiently probe target loci in multiple species. Consequently, more loci can be probed across species with a given number of baits. Using transcript sequences of 24 apoid wasps (Hymenoptera: Crabronidae, Sphecidae) from the 1KITE project and the gene models of Nasonia vitripennis , we inferred 57,650, 120-bp-long baits for capturing 378 coding sequence sections of 282 genes in apoid wasps. Illumina reduced-representation library sequencing confirmed successful enrichment of the target DNA when applying these baits to DNA of various apoid wasps. The designed baits furthermore enriched a major fraction of the target DNA in distantly related Hymenoptera, such as Formicidae and Chalcidoidea, highlighting the baits’ broad taxonomic applicability. The availability of baits with broad taxonomic applicability is of major interest in numerous disciplines, ranging from phylogenetics to biodiversity monitoring. We implemented our new approach in a software package, called BaitFisher, which is open source and freely available at https://github.com/cmayer/BaitFisher-package.git .

Description: Host-microbe associations underlie many key processes of host development, immunity, and life history. Yet, none of the current research on the central model species Caenorhabditis elegans considers the worm’s na...

Description: The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration.

Description: Liver disease due to alpha-1-antitrypsin deficiency (A1ATD) is associated with hepatic iron overload in a subgroup of patients. The underlying cause for this association is unknown. The aim of the present study was to define the genetics of this correlation and the effect of alpha-1-antitrypsin (A1AT) on the expression of the iron hormone hepcidin. Full exome and candidate gene sequencing were carried out in a family with A1ATD and hepatic iron overload. Regulation of hepcidin expression by A1AT was studied in primary murine hepatocytes. Cells co-transfected with hemojuvelin (HJV) and matriptase-2 (MT-2) were used as a model to investigate the molecular mechanism of this regulation. Observed familial clustering of hepatic iron overload with A1ATD suggests a genetic cause, but genotypes known to be associated with hemochromatosis were absent. Individuals homozygous for the A1AT Z-allele with environmental or genetic risk factors such as steatosis or heterozygosity for the HAMP non-sense mutation p.Arg59* presented with severe hepatic siderosis. In hepatocytes, A1AT induced hepcidin mRNA expression in a dose-dependent manner. Experiments in overexpressing cells show that A1AT reduces cleavage of the hepcidin inducing bone morphogenetic protein co-receptor HJV via inhibition of the membrane-bound serine protease MT-2. The acute-phase protein A1AT is an inducer of hepcidin expression. Through this mechanism, A1ATD could be a trigger of hepatic iron overload in genetically predisposed individuals or patients with environmental risk factors for hepatic siderosis.

Description: Background: How do very small animals with limited long-distance dispersal abilities move between locations, especially if they prefer ephemeral micro-habitats that are only available for short periods of time? The free-living model nematode Caenorhabditis elegans and several congeneric taxa appear to be common in such short-lived environments, for example decomposing fruits or other rotting plant material. Dispersal is usually assumed to depend on animal vectors, yet all current data is based on only a limited number of studies. In our project we performed three comprehensive field surveys on possible invertebrate vectors in North German locations containing populations of C. elegans and two related species, especially C. remanei, and combined these screens with an experimental analysis of persistence in one of the vector taxa. Results: Our field survey revealed that Caenorhabditis nematodes are commonly found in slugs, isopods, and chilopods, but are not present in the remaining taxonomic groups examined. Surprisingly, the nematodes were frequently isolated from the intestines of slugs, even if slugs were not collected in close association with suitable substrates for Caenorhabditis proliferation. This suggests that the nematodes are able to enter the slug intestines and persist for certain periods of time. Our experimental analysis confirmed the ability of C. elegans to invade slug intestines and subsequently be excreted alive with the slug feces, although only for short time periods under laboratory conditions. Conclusions: We conclude that three invertebrate taxonomic groups represent potential vectors of Caenorhabditis nematodes. The nematodes appear to have evolved specific adaptations to enter and persist in the harsh environment of slug intestines, possibly indicating first steps towards a parasitic life-style.

Description: Background: Chronic infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients, and a more complete understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics may provide a basis for improving intervention strategies. Here, we report the first genomic analysis of P. aeruginosa isolates sampled from Italian CF patients. Results: By genome sequencing of 26 isolates sampled over 19 years from four patients, we elucidated the within-host evolution of clonal lineages in each individual patient. Many of the identified mutations were located in pathoadaptive genes previously associated with host adaptation, and we correlated mutations with changes in CF-relevant phenotypes such as antibiotic resistance. In addition, the genomic analysis revealed that three patients shared the same clone. Furthermore, we compared the genomes of the Italian CF isolates to a panel of genome sequenced strains of P. aeruginosa from other countries. Isolates from two of the Italian lineages belonged to clonal complexes of P. aeruginosa that have previously been identified in Danish CF patients, and our genomic comparison showed that clonal isolates from the same country may be more distantly related than clonal isolates from different countries. Conclusions: This is the first whole-genome analysis of P. aeruginosa isolated from Italian CF patients, and together with both phenotypic and clinical information this dataset facilitates a more detailed understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics. We conclude that the evolution of the Italian lineages resembles what has been found in other countries.

Description: Background: The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions. However, cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly also high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing. Results: Here we describe a mammalian cell culture model, which correlates B lymphocyte migration of patient-derived MEC1 cells and asymmetric localization of fluorescently-tagged VANGL2. We show by live cell imaging that PCP proteins are polarized in MEC1 cells and that VANGL2 polarization is controlled by the same mechanism as in tissues i. e. it is dependent on casein kinase 1 activity. In addition, destruction of the actin cytoskeleton leads to migratory arrest and cell rounding while VANGL2-EGFP remains polarized suggesting that active PCP signaling visualized by polarized distribution of VANGL2 is a cause for and not a consequence of the asymmetric shape of a migrating cell. Conclusions: The presented imaging-based methodology allows overcoming limitations of earlier approaches to study the mammalian WNT/PCP pathway, which required in vivo models and analysis of complex tissues. Our system investigating PCP-like signaling on a single-cell level thus opens new possibilities for screening of compounds, which control asymmetric distribution of proteins in the PCP pathway.

Description: Background: A previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory. Results: The precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics. Conclusions: The results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.