Fine‐scale genetic structure in an eastern Alpine black grouse Tetrao tetrix metapopulation

Understanding genetic consequences of habitat fragmentation is crucial for the management and conservation of wildlife populations, especially in case of species sensitive to environmental changes and landscape alteration. In central Europe, the Alps are the core area of black grouse Tetrao tetrix distribution. There, black grouse dispersal is limited by high altitude mountain ridges and recent black grouse habitats are known to show some degree of natural fragmentation. Additionally, substantial anthropogenic fragmentation has occurred within the past ninety years. Facing losses of peripheral subpopulations and ongoing range contractions, we explored genetic variability and the fine‐scale genetic structure of the Alpine black grouse metapopulation at the easternmost fringe of the species’ Alpine range. Two hundred and fifty tissue samples and non‐invasive faecal and feather samples of eleven a priori defined subpopulations were used for genetic analysis based on nine microsatellite loci. Overall, eastern Alpine black grouse show similar amounts of genetic variation (H_O = 0.65, H_E = 0.66) to those found in more continuous populations like in Scandinavia. Despite of naturally and anthropogenically fragmented landscapes, genetic structuring was weak (global F_ST < 0.05), suggesting that the actual intensity of habitat fragmentation does not completely hamper dispersal, but probably restricts it to some extent. The most peripheral subpopulations at the edge of the species range show signs of genetic differentiation. The present study gives new insights into the population genetic structure of black grouse in the eastern Alps and provides a more fine‐scale view of genetic structure than previously available. Our findings will contribute to monitor the current and future status of the population under human pressures and to support supra‐regional land use planning as well as decision making processes in responsibilities of public administration.     

Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes

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Collection of airborne bacteria and yeast through water-based condensational growth

One limitation in air sampling of airborne microorganisms is their inactivation by forceful impaction and/or dehydration during the collection process. Proper inhalation risk assessments require proof of viability, as non-viable microorganisms cannot cause infectious diseases. In this study, laboratory-generated aerosols of a vegetative bacterium (E. coli) or yeast (S. kudriavzevii) were collected by a laminar-flow water-based condensational “growth tube collector (GTC),” and the GTC’s collection efficiencies were compared with those using an industry standard BioSampler. Collection efficiencies resulting from two types of collection media, phosphate-buffered saline (PBS) and nutrient media (Nutrient Broth, NB, for E. coli, and Yeast Tryptone Glucose Broth, YTGB, for S. kudriavzevii) were also assessed. Both the GTC and the BioSampler performed equally when PBS was used as the collection medium for E. coli, whereas more viable E. coli cells were collected in the GTC than the BioSampler with NB. For S. kudriavzevii, the GTC outperformed the BioSampler using either PBS or YTGB. This is likely because aerosolized E. coli cells can better survive impaction than S. kudriavzevii under the conditions used, and the BioSampler has a much higher collection efficiency for particles in the size range of single-celled E. coli than S. kudriavzevii. Moreover, the GTC had a detection limit one order of magnitude lower for yeast aerosols compared with that of the BioSampler. These results indicate that the GTC is a promising device for sampling viable aerosolized gram-negative bacteria and yeast, as it is less damaging to these types of microorganisms during the collection process.     

ATG16 mediates the autophagic degradation of the 19S proteasomal subunits PSMD1 and PSMD2

Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core autophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both, PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of autophagosomes, in wild-type cells. These puncta were absent in atg16￣ and atg9￣/16￣ cells and weaker and less frequent in atg9￣ cells, showing that ATG16 was crucial and the autophagic process important for their formation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in atg16￣ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2 positive puncta constitute autophagosomes. LysoTracker^® labeling and a proteolytic cleavage assay confirmed that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their enrichment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy and the ubiquitin proteasome system.     

RAD sequencing resolved phylogenetic relationships in European shrub willows (Salix L. subg. Chamaetia and subg. Vetrix) and revealed multiple evolution of dwarf shrubs

The large and diverse genus Salix L. is of particular interest for decades of biological research. However, despite the morphological plasticity, the reconstruction of phylogenetic relationships was so far hampered by the lack of informative molecular markers. Infrageneric classification based on morphology separates dwarf shrubs (subg. Chamaetia) and taller shrubs (subg. Vetrix), while previous phylogenetic studies placed species of these two subgenera just in one largely unresolved clade. Here we want to test the utility of genomic RAD sequencing markers for resolving relationships at different levels of divergence in Salix. Based on a sampling of 15 European species representing 13 sections of the two subgenera, we used five different RAD sequencing datasets generated by Ipyrad to conduct phylogenetic analyses. Additionally we reconstructed the evolution of growth form and analyzed the genetic composition of the whole clade. The results showed fully resolved trees in both ML and BI analysis with high statistical support. The two subgenera Chamaetia and Vetrix were recognized as nonmonophyletic, which suggests that they should be merged. Within the Vetrix/Chamaetia clade, a division into three major subclades could be observed. All species were confirmed to be monophyletic. Based on our data, arctic‐alpine dwarf shrubs evolved four times independently. The structure analysis showed five mainly uniform genetic clusters which are congruent in sister relationships observed in the phylogenies. Our study confirmed RAD sequencing as a useful genomic tool for the reconstruction of relationships on different taxonomic levels in the genus Salix.     

siRNA release from gold nanoparticles by nanosecond pulsed laser irradiation and analysis of the involved temperature increase

Nanosecond pulsed laser irradiation can trigger a release of nucleic acids from gold nanoparticles, but the involved nanoeffects are not fully understood yet. Here we investigate the release of coumarin labeled siRNA from 15 to 30 nm gold particles after nanosecond pulsed laser irradiation. Temperatures in the particle and near the surface were calculated for the different radiant exposures. Upon irradiation with laser pulses of 4 nanosecond duration release started for both particle sizes at a calculated temperature increase of approximately 500 K. Maximum coumarin release was observed for 15 nm particles after irradiation with radiant exposure of 80 mJ cm^?2 and with 32 mJ cm^?2 for 30 nm particles. This corresponds to a temperature increase of 815 and 900 K, respectively. Our results show that the molecular release by nanosecond pulsed irradiation is based on a different mechanism compared to continuous or femtosecond irradiation. Local temperatures are considerably higher and it is expected that bubble formation plays a crucial role in release and damage to cellular structures.      

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.     

Functional assessment of hepatobiliary secretion by 11C-cholylsarcosine positron emission tomography

Positron emission tomography (PET) with ¹¹C-cholylsarcosine (¹¹C-CSar), a radiolabelled synthetic N-methylglycine (sarcosine) conjugate of cholic acid, is a novel molecular imaging technique that enables quantitative assessment of the individual transport steps involved in hepatic secretion of conjugated bile acids. Here, we present the method and discuss its potential clinical and scientific applications based on findings in the first human study of healthy subjects and patients with cholestasis. We also present a clinical example of a patient studied during and six months after an episode of drug-induced cholestatic liver injury.