Automated identification of reference genes based on RNA-seq data

Background

Gene expression analyses demand appropriate reference genes (RGs) for normalization, in order to obtain reliable assessments. Ideally, RG expression levels should remain constant in all cells, tissues or experimental conditions under study. Housekeeping genes traditionally fulfilled this requirement, but they have been reported to be less invariant than expected; therefore, RGs should be tested and validated for every particular situation. Microarray data have been used to propose new RGs, but only a limited set of model species and conditions are available; on the contrary, RNA-seq experiments are more and more frequent and constitute a new source of candidate RGs.

Results

An automated workflow based on mapped NGS reads has been constructed to obtain highly and invariantly expressed RGs based on a normalized expression in reads per mapped million and the coefficient of variation. This workflow has been tested with Roche/454 reads from reproductive tissues of olive tree (Olea europaea L.), as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana and three different human cancers (prostate, small-cell cancer lung and lung adenocarcinoma). Candidate RGs have been proposed for each species and many of them have been previously reported as RGs in literature. Experimental validation of significant RGs in olive tree is provided to support the algorithm.

Conclusion

Regardless sequencing technology, number of replicates, and library sizes, when RNA-seq experiments are designed and performed, the same datasets can be analyzed with our workflow to extract suitable RGs for subsequent PCR validation. Moreover, different subset of experimental conditions can provide different suitable RGs.

     

External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca²⁺ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca²⁺ regulation of PC2 channel function. In this study, we explored the effect of external Ca²⁺ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca²⁺ concentrations, and observed that the basal whole cell conductance in normal Ca²⁺(1.2 mM), decreased by 30.2% in zero (nominal) Ca²⁺ and conversely, increased by 38% in high external Ca²⁺(6.2 mM). The high Ca²⁺-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca²⁺ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca²⁺ sensing receptor (CaSR) was implicated in the external Ca²⁺ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca²⁺ under Ca²⁺-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca²⁺. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca²⁺-dependent manner. The data support a novel Ca²⁺ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.     

Prey preferences and recent changes in diet of a breeding population of the Northern Goshawk Accipiter gentilis in Southwestern Europe

Capsule: Northern Goshawk Accipiter gentilis diet has changed significantly since the 1980s, probably due to changes in populations of preferred prey species.

Aims and methods: To assess changes to the breeding season diet of the Northern Goshawk in southwest Europe over three decades. We examined prey remains at and around nests and assessed avian prey availability using point count and line transect surveys.

Results: During 2008–11, Goshawks mainly ate birds, with Feral Pigeons Columba livia f. domestica being the most important prey species. Goshawks preferred prey of 100?400?g and forest prey species to non-forest species. Goshawk diet has changed significantly over recent decades: 22% of current prey items belong to species that were not part of the diet in the 1980s. We suggest that these dietary changes reflect changes in the abundance of prey species of the preferred size caused by changes in land use leading to an increase in forest cover, new prey species colonization and changes in the abundance and management of domestic prey.

Conclusion: This study emphasizes that major transformations occurring in agroforestry systems are affecting the main preferred prey of important forest predators, which may have consequences for conservation of both the predators and their prey.     

A realistic meteorological assessment of perennial biofuel crop deployment: a Southern Great Plains perspective

Utility of perennial bioenergy crops (e.g., switchgrass and miscanthus) offers unique opportunities to transition toward a more sustainable energy pathway due to their reduced carbon footprint, averted competition with food crops, and ability to grow on abandoned and degraded farmlands. Studies that have examined biogeophysical impacts of these crops noted a positive feedback between near‐surface cooling and enhanced evapotranspiration (ET), but also potential unintended consequences of soil moisture and groundwater depletion. To better understand hydrometeorological effects of perennial bioenergy crop expansion, this study conducted high‐resolution (2‐km grid spacing) simulations with a state‐of‐the‐art atmospheric model (Weather Research and Forecasting system) dynamically coupled to a land surface model. We applied the modeling system over the Southern Plains of the United States during a normal precipitation year (2007) and a drought year (2011). By focusing the deployment of bioenergy cropping systems on marginal and abandoned farmland areas (to reduce the potential conflict with food systems), the research presented here is the first realistic examination of hydrometeorological impacts associated with perennial bioenergy crop expansion. Our results illustrate that the deployment of perennial bioenergy crops leads to widespread cooling (1–2 °C) that is largely driven by an enhanced reflection of shortwave radiation and, secondarily, due to an enhanced ET. Bioenergy crop deployment was shown to reduce the impacts of drought through simultaneous moistening and cooling of the near‐surface environment. However, simulated impacts on near‐surface cooling and ET were reduced during the drought relative to a normal precipitation year, revealing differential effects based on background environmental conditions. This study serves as a key step toward the assessment of hydroclimatic sustainability associated with perennial bioenergy crop expansion under diverse hydrometeorological conditions by highlighting the driving mechanisms and processes associated with this energy pathway.     

World without borders—genetic population structure of a highly migratory marine predator,the blue shark (Prionace glauca)

Highly migratory, cosmopolitan oceanic sharks often exhibit complex movement patterns influenced by ontogeny, reproduction, and feeding. These elusive species are particularly challenging to population genetic studies, as representative samples suitable for inferring genetic structure are difficult to obtain. Our study provides insights into the genetic population structure one of the most abundant and wide‐ranging oceanic shark species, the blue shark Prionace glauca, by sampling the least mobile component of the populations, i.e., young‐of‐year and small juveniles (<2 year; N = 348 individuals), at three reported nursery areas, namely, western Iberia, Azores, and South Africa. Samples were collected in two different time periods (2002–2008 and 2012–2015) and were screened at 12 nuclear microsatellites and at a 899‐bp fragment of the mitochondrial control region. Our results show temporally stable genetic homogeneity among the three Atlantic nurseries at both nuclear and mitochondrial markers, suggesting basin‐wide panmixia. In addition, comparison of mtDNA CR sequences from Atlantic and Indo‐Pacific locations also indicated genetic homogeneity and unrestricted female‐mediated gene flow between ocean basins. These results are discussed in light of the species' life history and ecology, but suggest that blue shark populations may be connected by gene flow at the global scale. The implications of the present findings to the management of this important fisheries resource are also discussed.     

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis

Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use ¹³C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of ¹³C-labeled carbon from diatom extracellular polymeric substances (¹³C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from ¹³C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more ¹³C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on ¹³C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.Subject terms: Microbial ecology, Fungal ecology, Microbiome, Biogeochemistry