Consideration of RNA secondary structure significantly improves likelihood-based estimates of phylogeny: examples from the bilateria

Sequences from ribosomal RNA (rRNA) genes have made a huge contribution to our current understanding of metazoan phylogeny and indeed the phylogeny of all of life. That said, some parts of this rRNA-based phylogeny remain unresolved. One approach to increase the resolution of these trees would be to use more appropriate models of sequence evolution in phylogenetic analysis. RNAs transcribed from rRNA genes have a complex secondary structure mediated by base pairing between sometimes distant regions of the rRNA molecule. The pairing between the stem nucleotides has important consequences for their evolution which differs from that of unpaired loop nucleotides. These differences in evolution should ideally be accounted for when using rRNA sequences for phylogeny estimation. We use a novel permutation approach to demonstrate the significant superiority of models of sequence evolution that allow stem and loop regions to evolve according to separate models and, in common with previous studies, we show that 16-state models that take base pairing of stems into account are significantly better than simpler, 4-state, single-nucleotide models. One of these 16-state models has been applied to the phylogeny of the Bilateria using small subunit rRNA (SSU) sequences. Our optimal tree largely echoes previous results based on SSU in particular supporting the tripartite Bilaterian tree of deuterostomes, lophotrochozoans, and ecdysozoans. There are also a number of differences, however, perhaps most important of which is the observation of a clade consisting of the gastrotrichs plus platyheminthes that is basal to all other lophotrochozoan taxa. Use of 16-state models also appears to reduce the Bayesian support given to certain biologically improbable groups found using standard 4-state models.     

Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

Nonlinear simulation of tumor necrosis,neo-vascularization and tissue invasion via an adaptive finite-element/level-set method

We present a multi-scale computer simulator of cancer progression at the tumoral level, from avascular stage growth, through the transition from avascular to vascular growth (neo-vascularization), and into the later stages of growth and invasion of normal tissue. We use continuum scale reaction-diffusion equations for the growth component of the model, and a combined continuum-discrete model for the angiogenesis component. We use the level set method for describing complex topological changes observed during growth such as tumor splitting and reconnection, and capture of healthy tissue inside the tumor. We use an adaptive, unstructured finite element mesh that allows for finely resolving important regions of the computational domain such as the necrotic rim, the tumor interface and around the capillary sprouts. We present full nonlinear, two-dimensional simulations, showing the potential of our virtual cancer simulator. We use microphysical parameters characterizing malignant glioma cells, obtained from recent in vitro experiments from our lab and from clinical data, and provide insight into the mechanisms leading to infiltration of the brain by the cancer cells. The results indicate that diffusional instability of tumor mass growth and the complex interplay with the developing neo-vasculature may be powerful mechanisms for tissue invasion.     

Specificity in the coacervation of tropoelastin: solvent exposed lysines

Tropoelastin protein monomers associate by coacervation and are cross-linked in vivo to form elastin macro-assemblies. We provide evidence for specific protein domain contact points between tropoelastin monomers during association by coacervation. The homobifunctional cross-linker bis(sulfosuccinimidyl) suberate served as a rapid reporter of adjacent lysines and preferentially exposed domains. Intact cross-linked peptide pairs were identified after protease digestion and high-resolution electrospray mass spectrometry followed by MS/MS sequencing. Mapping of the assigned sequences indicated that the region in the monomer spanning domains 19-25 was readily accessible to solvent and enriched in cross-linking. Domains 12 and 36 were also prevalent, where these two regions were not previously thought to play a major role in the formation of mature elastin. A specificity for particular lysines allowed for the construction of a model for the first close contacts between domains and the first detailed study of the cross-linking of tropoelastin.     

Prefrontal cortex activity related to abstract response strategies

Many monkeys adopt abstract response strategies as they learn to map visual symbols to responses by trial and error. According to the repeat-stay strategy, if a symbol repeats from a previous, successful trial, the monkeys should stay with their most recent response choice. According to the change-shift strategy, if the symbol changes, the monkeys should shift to a different choice. We recorded the activity of prefrontal cortex neurons while monkeys chose responses according to these two strategies. Many neurons had activity selective for the strategy used. In a subsequent block of trials, the monkeys learned fixed stimulus-response mappings with the same stimuli. Some neurons had activity selective for choosing responses based on fixed mappings, others for choosing based on abstract strategies. These findings indicate that the prefrontal cortex contributes to the implementation of the abstract response strategies that monkeys use during trial-and-error learning.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.     

Y-chromosome and mitochondrial DNA studies on the population structure of the Christmas Island community

Christmas Island is a remote Australian territory located close to the main Indonesian island of Java. Y-chromosome and mitochondrial DNA (mtDNA) markers were used to investigate the genetic structure of the population, which comprises communities of mixed ethnic origin. Analysis of 12 Y-chromosome biallelic polymorphisms revealed a high level of gene diversity and haplotype frequencies that were consistent with source populations in southern China and Southeast Asia. mtDNA hypervariable segment I (HVS-I) sequences displayed high levels of haplotype diversity and nucleotide diversity that were comparable to various Asian populations. Genetic distances revealed extremely low mtDNA differentiation among Christmas Islanders and Asian populations. This was supported by the relatively high proportion of sequence types shared among these populations. The most common mtDNA haplogroups were M* and B, followed by D and F, which are prevalent in East/Southeast Asia. Christmas Islanders of European descent were characterized by the Eurasian haplogroup R*, and a limited degree of admixture was observed. In general, analysis of the genetic data indicated population affinities to southern Chinese (in particular from the Yunnan Province) and Southeast Asia (Thailand, Malaysia, and Cambodia), which was consistent with historical records of settlement. The combined use of these different marker systems provides a useful and appropriate model for the study of contemporary populations derived from different ethnic origins.