Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus

Imaging dielectric relaxation in nanostructured polymers by frequency modulation electrostatic force microscopy

We have developed a method for imaging the temperature-frequency dependence of the dynamics of nanostructured polymer films with spatial resolution. This method provides images with dielectric compositional contrast well decoupled from topography. Using frequency-modulation electrostatic-force-microscopy, we probe the local frequency-dependent (0.1–100 Hz) dielectric response through measurement of the amplitude and phase of the force gradient in response to an oscillating applied electric field. When the phase is imaged at fixed frequency, it reveals the spatial variation in dielectric losses, i.e., the spatial variation in molecular/dipolar dynamics, with 40 nm lateral resolution. This is demonstrated by using as a model system; a phase separated polystyrene/polyvinyl-acetate (PVAc) blend. We show that nanoscale dynamic domains of PVAc are clearly identifiable in phase images as those which light-up in a band of temperature, reflecting the variations in the molecular/dipolar dynamics approaching the glass transition temperature of PVAc

Bacteriën en schimmels op melkveebedrijf in beeld

Environmental Biolog

Microbiota in dung and milk differ between organic and conventional dairy farms

Organic farming is increasingly promoted as a means to reduce the environmental impact of artificial fertilizers, pesticides, herbicides, and antibiotics in conventional dairy systems. These factors potentially affect the microbial communities of the production stages (soil, silage, dung, and milk) of the entire farm cycle. However, understanding whether the microbiota representative of different production stages reflects different agricultural practices - such as conventional versus organic farming - is unknown. Furthermore, the translocation of the microbial community across production stages is scarcely studied. We sequenced the microbial communities of soil, silage, dung, and milk samples from organic and conventional dairy farms in the Netherlands. We found that community structure of soil fungi and bacteria significantly differed among soil types, but not between organic versus conventional farming systems. The microbial communities of silage also did not differ among conventional and organic systems. Nevertheless, the dung microbiota of cows and the fungal communities in the milk were significantly structured by agricultural practice. We conclude that, while the production stages of dairy farms seem to be disconnected in terms of microbial transfer, certain practices specific for each agricultural system, such as the content of diet and the use of antibiotics, are potential drivers of shifts in the cow's microbiota, including the milk produced. This may reflect differences in farm animal health and quality of dairy products depending on farming practices.Environmental Biolog

Relevant factors for the optimal duration of extended endocrine therapy in early breast cancer

Purpose: For postmenopausal patients with hormone receptor-positive early breast cancer, the optimal subgroup and duration of extended endocrine therapy is not clear yet. The aim of this study using the IDEAL patient cohort was to identify a subgroup for which longer (5 years) extended therapy is beneficial over shorter (2.5 years) extended endocrine therapy. Methods: In the IDEAL trial, 1824 patients who completed 5 years of adjuvant endocrine therapy (either 5 years of tamoxifen (12%), 5 years of an AI (29%), or a sequential strategy of both (59%)) were randomized between either 2.5 or 5 years of extended letrozole. For each prior therapy subgroup, the value of longer therapy was assessed for both node-negative and node-positive patients using Kaplan Meier and Cox regression survival analyses. Results: In node-positive patients, there was a significant benefit of 5 years (over 2.5 years) of extended therapy (disease-free survival (DFS) HR 0.67, p = 0.03, 95% CI 0.47–0.96). This effect was only observed in patients who were treated initially with a sequential scheme (DFS HR 0.60, p = 0.03, 95% CI 0.38–0.95). In all other subgroups, there was no significant benefit of longer extended therapy. Similar results were found in patients who were randomized for their initial adjuvant therapy in the TEAM trial (DFS HR 0.37, p = 0.07, 95% CI 0.13–1.06), although this additional analysis was underpowered for definite conclusions. Conclusions: This study suggests that node-positive patients could benefit from longer extended endocrine therapy, although this effect appears isol