Context-Dependent Substitution Dynamics in Plastid DNA Across a Wide Range of Taxonomic Groups.

The influence of neighboring base composition, or context, on substitution bias at fourfold degenerate coding sites and in intergenic regions in plastid DNA is compared across the angiosperms, gymnosperms, ferns, liverworts, chlorophytes, stramenopiles and rhodophytes. An influence of flanking base G + C content on the relative rates of transitions and transversions is observed in all lineages and extends up to four nucleotides from the site of substitution in some. Despite finding context effects in all lineages, significant differences were observed between lineages. Overall, the data suggest that context is a general factor affecting mutation bias in plastid DNA but that the dynamics of the influence have evolved over time. It is also shown that, although there are similar effects of context on substitution bias at fourfold degenerate coding sites and at sites within intergenic regions, there are also small but significant differences, suggesting that there could be some selection on some of these sites and that there could be some difference in the mutation and/or repair process between coding and noncoding DNA.

Context-Dependent Mutation Dynamics, Not Selection, Explains the Codon Usage Bias of Most Angiosperm Chloroplast Genes.

Two competing proposals about the degree to which selection affects codon usage of angiosperm chloroplast genes are examined. The first, based on observations that codon usage does not match expectations under the naïve assumption that base composition will be identical at all neutral sites, is that selection plays a significant role. The second is that codon usage is determined almost solely by mutation bias and drift, with selection influencing only one or two highly expressed genes, in particular psbA. First it is shown that, as a result of an influence of neighboring base composition on mutation dynamics, compositional biases are expected to be widely divergent at different sites in the absence of selection. The observed mutation properties are then used to predict expected neutral codon usage biases and to show that observed deviations from the naïve expectations are in fact expected given the context-dependent mutational dynamics. It is also shown that there is a match between the observed and expected codon usage when context effects are taken into consideration, with psbA being a notable exception. Overall, the data support the model that selection is not a widespread factor affecting the codon usage of angiosperm chloroplast genes and highlight the need to have an accurate model of mutational dynamics.

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene.

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.

Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing.

CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs-coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1 h after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. OBJECTIVES: We sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. METHODS: Bleomycin-induced lung fibrosis in wild-type and miR-155-/- mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors. RESULTS: miR-155-/- mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-ß production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-/- fibroblasts. CONCLUSIONS: We describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF.

Gustatory receptor neurons in Manduca sexta contain a TrpA1-dependent signaling pathway that integrates taste and temperature.

Temperature modulates the peripheral taste response of many animals, in part by activating transient receptor potential (Trp) cation channels. We hypothesized that temperature would also modulate peripheral taste responses in larval Manduca sexta. We recorded excitatory responses of the lateral and medial styloconic sensilla to chemical stimuli at 14, 22, and 30 °C. The excitatory responses to 5 chemical stimuli-a salt (KCl), 3 sugars (sucrose, glucose, and inositol) and an alkaloid (caffeine)-were unaffected by temperature. In contrast, the excitatory response to the aversive compound, aristolochic acid (AA), increased robustly with temperature. Next, we asked whether TrpA1 mediates the thermally dependent taste response to AA. To this end, we 1) identified a TrpA1 gene in M. sexta; 2) demonstrated expression of TrpA1 in the lateral and medial styloconic sensilla; 3) determined that 2 TrpA1 antagonists (HC-030031 and mecamylamine) inhibit the taste response to AA, but not caffeine; and then 4) established that the thermal dependence of the taste response to AA is blocked by HC-030031. Taken together, our results indicate that TrpA1 serves as a molecular integrator of taste and temperature in M. sexta.

Evolutionary history of relict Congeria (Bivalvia: Dreissenidae): unearthing the subterranean biodiversity of the Dinaric Karst.

BACKGROUND: Patterns of biodiversity in the subterranean realm are typically different from those encountered on the Earth's surface. The Dinaric karst of Croatia, Slovenia and Bosnia and Herzegovina is a global hotspot of subterranean biodiversity. How this was achieved and why this is so remain largely unresolved despite a long tradition of research. To obtain insights into the colonisation of the Dinaric Karst and the effects of the subterranean realm on its inhabitants, we studied the tertiary relict Congeria, a unique cave-dwelling bivalve (Dreissenidae), using a combination of biogeographical, molecular, morphological, and paleontological information. RESULTS: Phylogenetic and molecular clock analyses using both nuclear and mitochondrial markers have shown that the surviving Congeria lineage has actually split into three distinct species, i.e., C. kusceri, C. jalzici sp. nov. and C. mulaomerovici sp. nov., by vicariant processes in the late Miocene and Pliocene. Despite millions of years of independent evolution, analyses have demonstrated a great deal of shell similarity between modern Congeria species, although slight differences in hinge plate structure have enabled the description of the two new species. Ancestral plesiomorphic shell forms seem to have been conserved during the processes of cave colonisation and subsequent lineage isolation. In contrast, shell morphology is divergent within one of the lineages, probably due to microhabitat differences. CONCLUSIONS: Following the turbulent evolution of the Dreissenidae during the Tertiary and major radiations in Lake Pannon, species of Congeria went extinct. One lineage survived, however, by adopting a unique life history strategy that suited it to the underground environment. In light of our new data, an alternative scenario for its colonisation of the karst is proposed. The extant Congeria comprises three sister species that, to date, have only been found to live in 15 caves in the Dinaric karst. Inter-specific morphological stasis and intra-specific ecophenotypic plasticity of the congerid shell demonstrate the contrasting ways in which evolution in the underground environments shapes its inhabitants.

Do Noncoding and Coding Sites in Angiosperm Chloroplast DNA Have Different Mutation Processes?

Fourfold degenerate sites within coding regions and intergenic sites have both been used as estimates of neutral evolution. In chloroplast DNA, the pattern of substitution at intergenic sites is strongly dependent on the composition of the surrounding hexanucleotide composed of the three base pairs on each side, which suggests that the mutation process is highly context-dependent in this genome. This study examines the context-dependency of substitutions at fourfold degenerate sites in protein-coding regions and compares the pattern to what has been observed at intergenic sites. Overall, there is strong similarity between the two types of sites, but there are some intriguing differences. One of these is that substitutions of G and C are significantly higher at fourfold degenerate sites across a range of contexts. In fact, A → T and T → A substitutions are the only substitution types that occur at a lower rate at fourfold degenerate sites. The data are not consistent with selective constraints being responsible for the difference in substitution patterns between intergenic and fourfold degenerate sites. Rather, it is suggested that the difference may be a result of different epigenetic modifications that result in slightly different mutation patterns in coding and intergenic DNA.

Context and Mutation in Gymnosperm Chloroplast DNA.

Mutations and subsequent repair processes are known to be strongly context-dependent in the flowering-plant chloroplast genome. At least six flanking bases, three on each side, can have an influence on the relative rates of different types of mutation at any given site. In this analysis, examine context and substitution at noncoding and fourfold degenerate coding sites in gymnosperm DNA. The sequences are analyzed in sets of three, allowing the inference of the substitution direction and the generation of context-dependent rate matrices. The size of the dataset limits the analysis to the tetranucleotide context of the sites, but the evidence shows that there are significant contextual effects, with patterns that are similar to those observed in angiosperms. These effects most likely represent an influence on the underlying mutation/repair dynamics. The data extend the plastome lineages that feature very complex patterns of mutation, which can have significant effects on the evolutionary dynamics of the chloroplast genome.

Evidence for Strand Asymmetry in Different Plastid Genomes.

A common genome composition pattern in eubacteria is an asymmetry between the leading and lagging strands resulting in opposite skew patterns in the two replichores that lie between the origin and terminus of replication. Although this pattern has been reported for a couple of isolated plastid genomes, it is not clear how widespread it is overall in this chromosome. Using a random walk approach, we examine plastid genomes outside of the land plants, which are excluded since they are known not to initiate replication at a single site, for such a pattern of asymmetry. Although it is not a common feature, we find that it is detectable in the plastid genome of species from several diverse lineages. The euglenozoa in particular show a strong skew pattern as do several rhodophytes. There is a weaker pattern in some chlorophytes but it is not apparent in other lineages. The ramifications of this for analyses of plastid evolution are discussed.

Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference.

BACKGROUND: Insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial. RESULTS: Here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues. CONCLUSIONS: We report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

Substitution rate heterogeneity across hexanucleotide contexts in noncoding chloroplast DNA.

Substitutions between closely related noncoding chloroplast DNA sequences are studied with respect to the composition of the 3 bases on each side of the substitution, that is the hexanucleotide context. There is about 100-fold variation in rate, among the contexts, particularly on substitutions of A and T. Rate heterogeneity of transitions differs from that of transversions, resulting in a more than 200-fold variation in the transitions: transversion bias. The data are consistent with a CpG effect, and it is shown that both the A + T content and the arrangement of purines/pyrimidines along the same DNA strand are correlated with rate variation. Expected equilibrium A + T content ranges from 36.4% to 82.8% across contexts, while G-C skew ranges from -77.4 to 72.2 and A-T skew ranges from -63.9 to 68.2. The predicted equilibria are associated with specific features of the content of the hexanucleotide context, and also show close agreement with the observed context-dependent compositions. Finally, by controlling for the content of nucleotides closer to the substitution site, it is shown that both the third and fourth nucleotide removed on each side of the substitution directly influence substitution dynamics at that site. Overall, the results demonstrate that noncoding sites in different contexts are evolving along very different evolutionary trajectories and that substitution dynamics are far more complex than typically assumed. This has important implications for a number of types of sequence analysis, particularly analyses of natural selection, and the context-dependent substitution matrices developed here can be applied in future analyses.

Perspectives on Using Fast-Dissolving Paracetamol for Mild-to-Moderate Pain Management in Elderly or Diabetic Patients with Delayed Gastric Emptying Rates: An Exploratory Study.

Purpose: Pain is considered a major clinical and socioeconomic problem worldwide. Delayed gastric emptying rates allegedly influence the suitability of pain-relief medications in patient populations such as the elderly and individuals with diabetes. Faster pain relief was reportedly achieved by using a fast-dissolving paracetamol (FD-APAP) formulation. This study aims to gain insights to healthcare professionals' (HCPs) perspectives towards using FD-APAP in addressing barriers of pain relief in patient populations susceptible to delayed gastric emptying rates. Patients and Methods: A two-step modified Delphi consensus study was conducted among a panel of 10 general practitioners (GPs) and four endocrinologists. The first step comprises in-depth discussions around evidence relevant to FD-APAP for pain management in patient populations with delayed gastric emptying rates (elderly/diabetics). The second step (a survey) was based on the summarized input from the first step. Consensus was accepted upon ≥70% agreement. Perspectives on pain management by FD-APAP for elderly patients were explored among GPs, while those for diabetic patients were explored among GPs and endocrinologists. Results: Consensus was achieved, whereby all panel members found FD-APAP to be favorable and relevant for pain management in the elderly and the diabetic patient populations. GPs additionally raised the consensus on the importance of considering comorbidities associated with diabetes while having minimal complications during pain management. Conclusion: The panel found the faster disintegration and absorption of FD-APAP relevant and useful for patients with delayed gastric emptying, wherein, pain in the elderly and those with diabetes could be treated effectively with limited side-effects.

The origins, relationships, evolution and conservation of the weirdest marine bivalves: The watering pot shells. A review.

The fossil record shows that the two clavagelloid or watering pot families evolved at different times, the Clavagellidae first in the late Mesozoic (100-66mya), the Penicillidae later in the Cenozoic (33-23mya)-the former originally with, thus, a near-global Tethyan distribution, the latter restricted to the Indo-West Pacific. Representatives of the two clavagelloid families, moreover, have wholly different adventitious tube/crypt structures and, thus, methods of formation suggesting that evolutionary experiments have been undertaken to achieve such radical architectural novelties. This has resulted in one of the most surprising examples of convergent evolution in the Bivalvia. But, what were the ancestors of the Clavagelloidea? The shell and internal morphology of representatives of the three recognized genera of the Lyonsiidae, that is, Lyonsia, Entodesma and Mytilimeria, are described. Species of the latter two genera are highly specialized epibenthic, byssate, nestlers and embedded symbionts of ascidian colonies and sponges, respectively. Species of Lyonsia, however, are mostly shallow endobenthic burrowers. On the basis of these studies, it is concluded that species of Lyonsia can be regarded as representative of the ancestral watering pot (Clavagelloidea) condition. Evidence for this conclusion include the mineralogy, characteristics and ligament structure of the shell and features of the anatomy, importantly the modification of the vestigial pedal retractor muscles to form simple (Clavagellidae) and more complex (Penicillidae) proprioreceptors. Such an anatomy-based conclusion is supported to some extent by DNA analyses of representatives of the Lyonsiidae and the two constituent families of the Clavagelloidea. To some extent because all clavagelloids are exceedingly rare hindering such analyses. Such rarity, however, also argues for the strict conservation of all the species of the Clavagelloidea.

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Predatory marine bivalves: A review.

Most bivalves are suspension feeders. On the deep sea floor, however, some are predators, typically of meiobenthic crustaceans: copepods, cumaceans and ostracods. Propeamusiid scallops are one such group of predators. The largest numbers of predators, however, belong to the bivalve subclass Anomalodesmata and constitute, as currently recognised, some 500 species belonging principally to the Verticordioidea (120), Poromyoidea (75) and Cuspidarioidea (304) with four, two and four constituent families, respectively. A further family, the Parilimyidae, is considered to be derived from the Pholadomyoidea-the anomalodesmatan ancestor. These, generally small (<60mm shell length), nacreous and thin-shelled predators share many anatomical features that formerly allowed them to be collectively classified as the Septibranchia. Although this name is now rarely used, it refers to their possession of a ctenidially-derived septum in the mantle cavity and functioning in prey capture. Generally, there is a trend, possibly evolutionary, from a typical bivalve ctenidium (Parilimyidae and some Verticordioidea) to a complete septum (other Verticordioidea, Poromyoidea and Cuspidarioidea). In addition, the inhalant siphon, foot, labial palps, mouth and its lips play a role in prey capture, and ingestion. Similarly, the stomach is modified to digest such, typically chitinous, ingested prey. Most septibranchs are either consecutive or simultaneous hermaphrodites with self-fertilisation possibly usual and with some evidence in a few of larval brooding. Notwithstanding, the deep sea septibranch species are poorly studied with virtually nothing being known about their wider distributions, ecology, detailed reproductive strategies and life history traits.

Mass spawning by the date mussel Lithophaga lithophaga.

Lithophaga lithophaga is one of the commonest bivalves in the Mediterranean Sea and is present in almost every subtidal calcareous rock. Its reproductive cycle is known only from laboratory studies. Herein, we present data on the species reproductive activities based on localised but mass synchronized spawning events. The species reproduces at the end of the northern hemisphere summer and the majority of significant spawning events occur during the period between full moon and its last quarter. Calm seas are an important pre-requisite for the development of such co-ordinated mass spawning events. 'Gamete to gamete' induction seems to be the most likely proximate cue in synchronising gamete release. Spawning begins with a few individuals but spreads progressively along the coastline. In observed situations, reproductive waves finally affect between 10 and >400 m of coastline from 0 to 10 m depth and last longer than three days. In the reproductive zone, dense gamete clouds reduce visibility to zero over tens of metres along the shallow sea bed. No spawning events of such dimensions have been reported upon before for any bivalve.

Separating the effects of mutation and selection in producing DNA skew in bacterial chromosomes.

BACKGROUND: Many bacterial chromosomes display nucleotide asymmetry, or skew, between the leading and lagging strands of replication. Mutational differences between these strands result in an overall pattern of skew that is centered about the origin of replication. Such a pattern could also arise from selection coupled with a bias for genes coded on the leading strand. The relative contributions of selection and mutation in producing compositional skew are largely unknown. RESULTS: We describe a model to quantify the contribution of mutational differences between the leading and lagging strands in producing replication-induced skew. When the origin and terminus of replication are known, the model can be used to estimate the relative accumulation of G over C and of A over T on the leading strand due to replication effects in a chromosome with bidirectional replication arms. The model may also be implemented in a maximum likelihood framework to estimate the locations of origin and terminus. We find that our estimations for the origin and terminus agree very well with the location of genes that are thought to be associated with the replication origin. This indicates that our model provides an accurate, objective method of determining the replication arms and also provides support for the hypothesis that these genes represent an ancestral cluster of origin-associated genes. CONCLUSION: The model has several advantages over other methods of analyzing genome skew. First, it quantifies the role of mutation in generating skew so that its effect on composition, for example codon bias, can be assessed. Second, it provides an objective method for locating origin and terminus, one that is based on chromosome-wide accumulation of leading vs lagging strand nucleotide differences. Finally, the model has the potential to be utilized in a maximum likelihood framework in order to analyze the effect of chromosome rearrangements on nucleotide composition.

Variation in mutation dynamics across the maize genome as a function of regional and flanking base composition.

We examine variation in mutation dynamics across a single genome (Zea mays ssp. mays) in relation to regional and flanking base composition using a data set of 10,472 SNPs generated by resequencing 1776 transcribed regions. We report several relationships between flanking base composition and mutation pattern. The A + T content of the two sites immediately flanking the mutation site is correlated with rate, transition bias, and GC --> AT pressure. We also observe a significant CpG effect, or increase in transition rate at CpG sites. At the regional level we find that the strength of the CpG effect is correlated with regional A + T content, ranging from a 1.7-fold increase in transition rate in relatively G + C-rich regions to a 2.6-fold increase in A + T-rich regions. We also observe a relationship between locus A + T content and GC --> AT pressure. This regional effect is in opposition to the influence of the two immediate neighbors in that GC --> AT pressure increases with increasing locus A + T content but decreases with increasing flanking base A + T content and may represent a relationship between genome location and mutation bias. The data indicate multiple context effects on mutations, resulting in significant variation in mutation dynamics across the genome.