Speciation with gene flow in a narrow endemic West Virginia cave salamander (Gyrinophilus subterraneus)

Due to their limited geographic distributions and specialized ecologies, cave species are often highly endemic and can be especially vulnerable to habitat degradation within and surrounding the cave systems they inhabit. We investigated the evolutionary history of the West Virginia Spring Salamander (Gyrinophilus subterraneus), estimated the population trend from historic and current survey data, and assessed the current potential for water quality threats to the cave habitat. Our genomic data (mtDNA sequence and ddRADseq-derived SNPs) reveal two, distinct evolutionary lineages within General Davis Cave corresponding to G. subterraneus and its widely distributed sister species, Gyrinophilus porphyriticus, that are also differentiable based on morphological traits. Genomic models of evolutionary history strongly support asymmetric and continuous gene flow between the two lineages, and hybrid classification analyses identify only parental and first generation cross (F1) progeny. Collectively, these results point to a rare case of sympatric speciation occurring within the cave, leading to strong support for continuing to recognize G. subterraneus as a distinct and unique species. Due to its specialized habitat requirements, the complete distribution of G. subterraneus is unresolved, but using survey data in its type locality (and currently the only known occupied site), we find that the population within General Davis Cave has possibly declined over the last 45 years. Finally, our measures of cave and surface stream water quality did not reveal evidence of water quality impairment and provide important baselines for future monitoring. In addition, our unexpected finding of a hybrid zone and partial reproductive isolation between G. subterraneus and G. porphyriticus warrants further attention to better understand the evolutionary and conservation implications of occasional hybridization between the species.

     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

Endocytosis and Signaling during Development

The development of multicellular organisms relies on an intricate choreography of intercellular communication events that pattern the embryo and coordinate the formation of tissues and organs. It is therefore not surprising that developmental biology, especially using genetic model organisms, has contributed significantly to the discovery and functional dissection of the associated signal-transduction cascades. At the same time, biophysical, biochemical, and cell biological approaches have provided us with insights into the underlying cell biological machinery. Here we focus on how endocytic trafficking of signaling components (e.g., ligands or receptors) controls the generation, propagation, modulation, reception, and interpretation of developmental signals. A comprehensive enumeration of the links between endocytosis and signal transduction would exceed the limits of this review. We will instead use examples from different developmental pathways to conceptually illustrate the various functions provided by endocytic processes during key steps of intercellular signaling.The evolution of multicellular life introduced a division of labor between specialized cells, which strongly increased demand for intercellular communication both during development and homeostasis of the adult organism (Kaiser 2001). At the genomic level this is reflected by a dramatic expansion of the surface receptor signalome in all metazoan lineages (Ben-Shlomo et al. 2003). However, the idea that intercellular communication drives the organization and patterning of the embryo precedes the identification of the responsible molecules. Induction (i.e., the ability of one group of cells within a developing organism to influence the cell-fate choices), morphogenesis, and differentiation of other cell populations, was firmly established by the experiments of Spemann and Mangold (1924) and has become one of the most important concepts of developmental biology. The related concept of the morphogen, whereby a cell can identify its position within a tissue by using the local levels of a secreted molecule forming a concentration gradient as a proxy for its distance from the source, was famously illustrated by the “French flag Model” by Wolpert (1969).Since then, examples of developmental patterning events following these two paradigms have been identified in all developmental model systems, ranging from worms and flies to amphibians, fish, and mice, and even humans. Surprisingly, despite the huge variety in the eventual outcome of metazoan embryonic development, it turned out that most individual patterning events are performed by a restricted set of signal-transduction pathways that are used repeatedly and in varying cellular contexts (Pires-daSilva and Sommer 2003; Perrimon et al. 2012).Because animal embryos differ in shape and size, closely related pathways must function over similarly varying spatial and temporal scales, potentially even at successive developmental stages within the same embryo. To understand how the limited, intercellular signaling repertoire is modulated to accommodate the varying patterning needs arising within the different developmental contexts, it is necessary to study the signal-transduction machinery at the molecular level. In recent years, major progress has been made in understanding the mechanistic links between the protein trafficking machinery and the generation and interpretation of morphogenetic signals.Traditionally, endocytosis was seen as a means of removing activated receptors and their bound ligands from the surface of the signal-receiving cells, thereby terminating the signals. However, positive effects of endocytosis on signal transduction have recently been identified for many different pathways including, among others, receptor tyrosine kinases (RTKs), TGF-β, TNF-α, Toll-like receptor, Wnt, and Notch signal-transduction cascades (Miaczynska et al. 2004; Platta and Stenmark 2011). In many of these examples, endosomes act as platforms where the activated receptors can interact with specific downstream components of the signal-transduction machinery (Sadowski et al. 2009; Miaczynska and Bar-Sagi 2010). Trafficking of the receptors into and out of such endosomes may thus provide another tier for the regulation of the signaling output that allows temporal and spatial modulation of the signals independent of ligand presentation. In addition, the endocytic pathway has recently also become implicated in signaling events that precede the intracellular transduction of the signal. In this review, we therefore focus on how the endocytic machinery participates in the generation, propagation, reception, and interpretation of intercellular signals in the context of animal development.     

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTR^sev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTR^BD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTR^BD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTR^BD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTR^BD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTR^BD variants through multiple mechanisms. CFTR^BD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.     

Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.     

A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.