High-Throughput,Amplicon-Based Sequencing of the CREBBP Gene as a Tool to Develop a Universal Platform-Independent Assay

High-throughput sequencing technologies are widely used to analyse genomic variants or rare mutational events in different fields of genomic research, with a fast development of new or adapted platforms and technologies, enabling amplicon-based analysis of single target genes or even whole genome sequencing within a short period of time. Each sequencing platform is characterized by well-defined types of errors, resulting from different steps in the sequencing workflow. Here we describe a universal method to prepare amplicon libraries that can be used for sequencing on different high-throughput sequencing platforms. We have sequenced distinct exons of the CREB binding protein (CREBBP) gene and analysed the output resulting from three major deep-sequencing platforms. platform-specific errors were adjusted according to the result of sequence analysis from the remaining platforms. Additionally, bioinformatic methods are described to determine platform dependent errors. Summarizing the results we present a platform-independent cost-efficient and timesaving method that can be used as an alternative to commercially available sample-preparation kits.     

Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-κB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.     

Robust Fall Social Displays Predict Spring Reproductive Behavior in Brown‐Headed Cowbirds (Molothrus ater ater)

The ability to engage others in close proximity may be an essential component of social life and shapes the development of social skills. Variation in the willingness to initiate and sustain close interaction with conspecifics is known as sociability. The Brown‐headed Cowbird (Molothrus ater) uses an affiliative display called the head‐down to bring individuals into close proximity. During fall 2009, we manipulated a large flock of cowbirds in a fission–fusion perturbation and recorded the frequency of head‐downs and social approaches. During the fission–fusion perturbation, the rate of head‐downs remained both correlated and repeatable across perturbations. In spring 2010, we separated individuals into three aviaries, a high, intermediate, and low aviary, based on the frequency of head‐down displays they initiated during the previous fall 2009. When breeding, males in the high flock produced a higher number of songs within counter‐singing matches, and females laid more eggs in comparison with the other aviaries. These findings suggest that head‐down displays performed outside the breeding season may contribute to the development and maintenance of reproductive competence by providing intimate social interactions with others.     

Differences between species in seed bank and vegetation helps to hold functional diversity in a floodable Neotropical savanna

种子库和植被物种之间的差异有助于保持新热带稀树草原的功能多样性 本研究的目的是量化种子库和建立的植被对物种组成、功能组成和多样性的贡献,并讨论这些差异对冲积平原植物群落更新和稳定性的影响。我们在巴西潘塔纳尔湿地的一个周期性洪水泛滥的稀树大草原上,在旱季和雨季对分布在5个地点的25个地块(10 m × 1 m)的1.5 m高度的地面覆盖植被和种子库进行了采样。采用出苗法对土壤种子库进行了评价。研究结果表明,种子库物种具有赋予群落更新的特征,而植被则具有稳定性特征。种子库的功能丰富度高于植被,而功能均匀度则低于植被。由于种子库和植被的植物特征不同,使得具有不同功能稳定性和更新特性的物种得以共存,这将有助于维持植物的功能多样性。这可能会让植物种群在应对干旱、火灾和洪水等不同的环境选择时更具有弹性。     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Seed bank of seasonally flooded grassland: experimental simulation of flood and post-flood

Wetland seed banks comprise the propagules of plant species that have species-specific germination requirements for germination in either flooded or dry conditions. At the community level, wetland structure and succession during and after a seasonal flooding event depends upon the early life-history requirements of species, including germination under flooded and dry conditions. We examined the effects of simulated flood and post-flood scenarios on seedling emergence from a seed bank of seasonally flooded grassland in the Pantanal, Brazil. Field samplings were conducted in both wet and dry seasons, both of which were subject to flood and post-flood conditions. A total of 70 species emerged from the seed bank, dominated by Poaceae and Cyperaceae. Sixteen species were exclusive to the wet and one exclusive to the dry season. The richness of perennial species was higher under flood conditions, while the richness of annuals was greater post-flood. In general, the aquatic and amphibious species exhibited a significant germination response to flooding. Terrestrial species only germinated in post-flood conditions, with higher richness in the dry season. Four species had high seedling abundance in both treatments. The capacity of regeneration by seeds is high in these grasslands and can be increased by seasonal flooding and drawdown. In these seasonally flooded grasslands, we observed three main germination strategies: under flooded conditions, aquatic and amphibious species; post-flood conditions, an explosion of annual amphibious and terrestrial species; and in moist soil, perennial terrestrial species. The differential responses to flooding versus post-flood conditions help to maintain the structure and species richness in the community over time.     

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current