A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.     

Increasing Water Temperature Triggers Dominance of Small Freshwater Plankton

Climate change scenarios predict that lake water temperatures will increase up to 4°C and rainfall events will become more intense and frequent by the end of this century. Concurrently, supply of humic substances from terrestrial runoff is expected to increase, resulting in darker watercolor (“brownification”) of aquatic ecosystems. Using a multi-seasonal, low trophic state mesocosm experiment, we investigated how higher water temperature and brownification affect plankton community composition, phenology, and functioning. We tested the hypothesis that higher water temperature (+3°C) and brownification will, a) cause plankton community composition to shift toward small sized phytoplankton and cyanobacteria, and, b) extend the length of the growing season entailing higher phytoplankton production later in the season. We demonstrate that the 3°C increase of water temperature favored the growth of heterotrophic bacteria and small sized autotrophic picophytoplankton cells with significantly higher primary production during warmer fall periods. However, 3X darker water (effect of brownification) caused no significant changes in the plankton community composition or functioning relative to control conditions. Our findings reveal that increased temperature change plankton community structure by favoring smaller sized species proliferation (autotrophic phytoplankton and small size cladocerans), and increase primary productivity and community turnover. Finally, results of this multi-seasonal experiment suggest that warming by 3°C in aquatic ecosystems of low trophic state may cause planktonic food web functioning to become more dominated by fast growing, r-trait species (i.e., small sizes and rapid development).     

Congruence,but no cascade—Pelagic biodiversity across three trophic levels in Nordic lakes

Covariation in species richness and community structure across taxonomical groups (cross‐taxon congruence) has practical consequences for the identification of biodiversity surrogates and proxies, as well as theoretical ramifications for understanding the mechanisms maintaining and sustaining biodiversity. We found there to exist a high cross‐taxon congruence between phytoplankton, zooplankton, and fish in 73 large Scandinavian lakes across a 750 km longitudinal transect. The fraction of the total diversity variation explained by local environment alone was small for all trophic levels while a substantial fraction could be explained by spatial gradient variables. Almost half of the explained variation could not be resolved between local and spatial factors, possibly due to confounding issues between longitude and landscape productivity. There is strong consensus that the longitudinal gradient found in the regional fish community results from postglacial dispersal limitations, while there is much less evidence for the species richness and community structure gradients at lower trophic levels being directly affected by dispersal limitation over the same time scale. We found strong support for bidirectional interactions between fish and zooplankton species richness, while corresponding interactions between phytoplankton and zooplankton richness were much weaker. Both the weakening of the linkage at lower trophic levels and the bidirectional nature of the interaction indicates that the underlying mechanism must be qualitatively different from a trophic cascade.     

Oviposition-site shift in phytophagous mites reflects a trade-off between predator avoidance and rainstorm resistance

Predators can reduce prey population densities by driving them to undertake costly defences. Here, we report on a remarkable example of induced antipredator defence in spider mites that enhances the risk to rainstorms. Spider mites live on the undersides of host plant leaves and usually oviposit on the leaf undersurface. When they are threatened by predatory mites, they oviposit on three-dimensional webs to avoid egg predation, although the cost of ovipositing on webs has not yet been clearly determined. We prepared bean plants harbouring spider mite (Tetranychus kanzawai) eggs on either leaf surfaces or webs and exposed them to rainstorms outdoors. We found that fewer eggs remained on webs than on leaf surfaces. We then examined the synergistic effect of wind and rain by simulating both in the laboratory. We conclude that ovipositing on webs comes at a cost, as eggs are washed off the host plants by wind and rain. This may explain why spider mite populations decrease drastically in the rainy season, although they inhibit leaf undersides where they are not directly exposed to rainfall.     

Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER–Golgi traffic and macroautophagy

ER-derived COPII-coated vesicles are conventionally targeted to the Golgi. However, during cell stress these vesicles also become a membrane source for autophagosomes, distinct organelles that target cellular components for degradation. How the itinerary of COPII vesicles is coordinated on these pathways remains unknown. Phosphorylation of the COPII coat by casein kinase 1 (CK1), Hrr25, contributes to the directional delivery of ER-derived vesicles to the Golgi. CK1 family members are thought to be constitutively active kinases that are regulated through their subcellular localization. Instead, we show here that the Rab GTPase Ypt1/Rab1 binds and activates Hrr25/CK1δ to spatially regulate its kinase activity. Consistent with a role for COPII vesicles and Hrr25 in membrane traffic and autophagosome biogenesis, hrr25 mutants were defective in ER–Golgi traffic and macroautophagy. These studies are likely to serve as a paradigm for how CK1 kinases act in membrane traffic.     

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H⁺-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca²⁺ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.     

Human mesenchymal stromal cells preserve their stem features better when cultured in the Dulbecco's modified Eagle medium

Background aimsThe human mesenchymal stromal cell (hMSC), a type of adult stem cell with a fibroblast-like appearance, has the potential to differentiate along the mesenchymal lineage and also along other cell lineages. These abilities make hMSC a promising candidate for use in regenerative medicine. As the hMSC represents a very rare population in vivo, in vitro expansion is necessary for any clinical use. hMSC characterization is commonly carried out through the expression of specific markers and by the capability of differentiating toward at least adipo-, osteo- and chondrocytic lineages. Commitment processes also result in significant changes in the ultrastructure in order to acquire new functional abilities; however, few studies have dealt with the ultrastructural characteristics of hMSC according to the time of incubation and type of media.MethodsThe immunophenotype, functional characteristics and ultrastructural features of bone marrow (BM) hMSC cultured in two different media were investigated. The media chosen were Iscove's modified Dulbecco's medium (IMDM) and the Dulbecco's modified Eagle medium (DMEM). The latter has been recommended recently by two international transplantation and cytotherapy societies, the International Society of Cellular Therapy (ISCT) and European Group for Blood and Bone Marrow Transplantation (EBMT), for hMSC expansion for clinical applications.Results and ConclusionsThe present results indicate that culture conditions greatly influence hMSC ultrastructural features, proliferation, growth and differentiation. In particular, our findings demonstrate that DMEM preserves the hMSC stem features better. Furthermore, the results obtained in IMDM suggest that a small size does not always correlate with conditions of cell immaturity and a greater proliferative potential.     

Effects of waterborne Cu exposure in gilthead sea bream (Sparus aurata): a proteomic approach

Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein).