Predicting Dissolved Organic Matter Lability and Carbon Accumulation in Temperate Freshwater Ecosystems

Ecosystems - Dissolved organic matter (DOM) dynamics influence aquatic ecosystem metabolism with ecological and biogeochemical effects. During microbial degradation, certain DOM molecules...     

Connectivity,diversity, and hybridization between two endemic fish species (Percilia spp.) in a complex temperate landscape

The identification of closely related species with partially overlapping distributions is fundamental for effective conservation. Here we analyzed 28 sequenced microsatellites, mtDNA sequences, and morphological data, to describe the connectivity, genetic relationship, and distribution of Percilia gillissi and Percilia irwini, two endangered species inhabiting two contiguous watersheds in Chile (Itata and Biobío). We provide evidence of discordance in the spatial distribution of the two genomes (nuclear and mitochondrial). Three large clusters were identified with microsatellites, with one cluster straddling both watersheds. Three clusters were also evident in mtDNA with one cluster straddling both watersheds and the other two restricted to the Itata watershed’s northern reaches. Analyses of both microsatellite and mtDNA identified P. gillissi in the Itata watershed northern reaches and P. irwini in the Biobío watershed. Fish were detected in the Itata watershed that carried mtDNA characteristic of P. irwini but nuclear microsatellite profiles of P. gillissi suggesting an incomplete reproductive barrier between the species and connectivity between the watersheds. Additionally, fish were identified in the Itata northern reaches carrying mtDNA haplotypes sufficiently distinct from those of P. gillissi and P. irwini to suggest the existence of higher mtDNA diversity within P. gillissi than previously recognized. Finally, there was limited support for taxonomical classification based on morphological and meristic traits in this region.

     

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.