Using phylogenomics to resolve mega-families: An example from Compositae

Next-generation sequencing and phylogenomics hold great promise for elucidating complex relationships among large plant families. Here, we performed targeted capture of low copy sequences followed by next-generation sequencing on the Illumina platform in the large and diverse angiosperm family Compositae (Asteraceae). The family is monophyletic, based on morphology and molecular data, yet many areas of the phylogeny have unresolved polytomies and interpreting phylogenetic patterns has been historically difficult. In order to outline a method and provide a framework and for future phylogenetic studies in the Compositae, we sequenced 23 taxa from across the family in which the relationships were well established as well as a member of the sister family Calyceraceae. We generated nuclear data from 795 loci and assembled chloroplast genomes from off-target capture reads enabling the comparison of nuclear and chloroplast genomes for phylogenetic analyses. We also analyzed multi-copy nuclear genes in our data set using a clustering method during orthology detection, and we applied a network approach to these clusters—analyzing all related locus copies. Using these data, we produced hypotheses of phylogenetic relationships employing both a conservative (restricted to only loci with one copy per targeted locus) and a multigene approach (including all copies per targeted locus). The methods and bioinformatics workflow presented here provide a solid foundation for future work aimed at understanding gene family evolution in the Compositae as well as providing a model for phylogenomic analyses in other plant mega-families.     

Toll-like receptor expression in normal ovary and ovarian tumors

Recent studies have implicated inflammation in the initiation and progression of ovarian cancer, though the mechanisms underlying this effect are still not clear. Toll-like receptors (TLRs) allow immune cells to recognize pathogens and to trigger inflammatory responses. Tumor cell expression of TLRs can promote inflammation and cell survival in the tumor microenvironment. Here we sought to characterize the expression of TLRs in normal human ovaries, benign and malignant ovarian tumors from patients, and in established ovarian tumor cell lines. We report that TLR2, TLR3, TLR4, and TLR5 are strongly expressed on the surface epithelium of normal ovaries. In contrast to previous studies of uterus and endocervix, we found no cyclic variation in TLR expression occurred in murine ovaries. TLR2, TLR3, TLR4, and TLR5 are expressed in benign conditions, epithelial tumors, and in ovarian cancer cell lines. Variable expression of TLR6 and TLR8 was seen in benign and malignant epithelium of some patients, while expression of TLR1, TLR7, and TLR9 was weak. Normal and malignant ovarian stroma were negative for TLR expression. Vascular endothelial cells, macrophages, and occasional fibroblasts in tumors were positive. Functional activity for TLRs was demonstrated by stimulation of cell lines with specific ligands and subsequent activation and translocation of NFκB and release of the proinflammatory cytokines interleukin-6 and CCL-2. These studies demonstrate expression of multiple TLRs in the epithelium of normal ovaries and in ovarian tumor cells, and may indicate a mechanism by which epithelial tumors manipulate inflammatory pathways to facilitate tumor progression.     

Prediction of Pharmacokinetic Profile of Valsartan in Humans Based on in vitro Uptake‐Transport Data

The aim of this study was to evaluate a physiologically based pharmacokinetic (PBPK) model for predicting PK profiles in humans based on a model refined in rats and humans in vitro uptake‐transport data using valsartan as a probe substrate. Valsartan is eliminated unchanged, mostly through biliary excretion, both in humans and rats. It was, therefore, chosen as model compound to predict in vivo elimination based on in vitro hepatic uptake‐transport data using a fully mechanistic PBPK model. Plated rat and human hepatocytes, and cell lines overexpressing human OATP1B1 and OATP1B3 were used for in vitro uptake experiments. A mechanistic two‐compartment model was used to derive the active and passive transport parameters, namely uptake Michaelis–Menten parameters (V_max and K_m,u) together with passive diffusion (P_dif). These transport parameters were then used as input in a whole body physiologically based pharmacokinetic (PBPK) model. The uptake rate of valsartan was higher for rat hepatocytes (K_m,u=28.4±3.7 μM , V_max=1320±180 pmol/mg/min, and P_dif =1.21±0.42 μl/mg/min) compared to human hepatocytes (K_m,u=44.4±14.6 μM , V_max=304±85 pmol/mg/min, and P_dif=0.724±0.271 μl/mg/min). OATP1B1 and ‐1B3 parameters were correlated to human hepatocyte data, using experimentally established relative activity factors (RAF). Resulting PBPK simulations were compared for plasma‐ (humans and rats) and bile‐ (rats) concentration–time profiles following iv bolus administration of valsartan. Plasma clearances (CL_P) for rats and humans were predicted within twofold relative to predictions based on respective in vitro data. The simulations were extended to simulate the impact of either OATP1B1 or ‐1B3 inhibition on plasma profile. The limited data set indicates that the mechanistic model allowed for accurate evaluation of in vitro transport data; and the resulting hepatic uptake transport kinetic parameters enabled the prediction of in vivo PK profiles and plasma clearances, using PBPK modelling. Moreover, the interspecies difference in elimination rate observed in vivo was correctly reflected in the transport parameters determined in vitro.     

Ecological factors influence population genetic structure of European grey wolves

Although the mechanisms controlling gene flow among populations are particularly important for evolutionary processes, they are still poorly understood, especially in the case of large carnivoran mammals with extensive continuous distributions. We studied the question of factors affecting population genetic structure in the grey wolf, Canis lupus, one of the most mobile terrestrial carnivores. We analysed variability in mitochondrial DNA and 14 microsatellite loci for a sample of 643 individuals from 59 localities representing most of the continuous wolf range in Eastern Europe. We tested an array of geographical, historical and ecological factors to check whether they may explain genetic differentiation among local wolf populations. We showed that wolf populations in Eastern Europe displayed nonrandom spatial genetic structure in the absence of obvious physical barriers to movement. Neither topographic barriers nor past fragmentation could explain spatial genetic structure. However, we found that the genetic differentiation among local populations was correlated with climate, habitat types, and wolf diet composition. This result shows that ecological processes may strongly influence the amount of gene flow among populations. We suggest natal-habitat-biased dispersal as an underlying mechanism linking population ecology with population genetic structure.     

Individual advantages to ecological specialization: insights on cognitive constraints from three conspecific taxa

The information-processing hypothesis (IPH) posits that specialist herbivores should make host-associated decisions more effectively than generalists and thus enjoy associated fitness advantages that may help explain the evolutionary prevalence of host-specific insects. This is because generalists must evaluate a greater diversity of host plants/cues than specialists and thus face a cognitive challenge that is predicted to constrain the efficiency and accuracy of their choices. Here, we present the first individual-level evaluation of this hypothesis. This involved experimentally quantifying the specificity, efficiency, and accuracy of host selection, as both larvae and adults, for many individuals representing each of three 'host forms' of Neochlamisus bebbianae leaf beetles. These experiments provided several significant findings: host forms differed in larval specificity, with the more specialized host forms more efficiently and accurately selecting optimal hosts as both larvae and adults. Positive correlations between larval specificity and both efficiency and accuracy across test individuals provided the most direct evidence to date for a biological association between these variables. Our results thus provide strong and consistent support for the IPH at the level of both populations and individuals. Because individual N. bebbianae make many host-associated decisions in nature, our results suggest that cognitive constraints may play a major role in the evolutionary dynamics of ongoing ecological specialization and diversification in this species.     

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinson''s disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.