Accuracy in Near-Perfect Virus Phylogenies.

Phylogenetic trees from real-world data often include short edges with very few substitutions per site, which can lead to partially resolved trees and poor accuracy. Theory indicates that the number of sites needed to accurately reconstruct a fully resolved tree grows at a rate proportional to the inverse square of the length of the shortest edge. However, when inferred trees are partially resolved due to short edges, "accuracy" should be defined as the rate of discovering false splits (clades on a rooted tree) relative to the actual number found. Thus, accuracy can be high even if short edges are common. Specifically, in a "near-perfect" parameter space in which trees are large, the tree length $\xi$ (the sum of all edge lengths) is small, and rate variation is minimal, the expected false positive rate is less than $\xi/3$; the exact value depends on tree shape and sequence length. This expected false positive rate is far below the false negative rate for small $\xi$ and often well below 5% even when some assumptions are relaxed. We show this result analytically for maximum parsimony and explore its extension to maximum likelihood using theory and simulations. For hypothesis testing, we show that measures of split "support" that rely on bootstrap resampling consistently imply weaker support than that implied by the false positive rates in near-perfect trees. The near-perfect parameter space closely fits several empirical studies of human virus diversification during outbreaks and epidemics, including Ebolavirus, Zika virus, and SARS-CoV-2, reflecting low substitution rates relative to high transmission/sampling rates in these viruses.[Ebolavirus; epidemic; HIV; homoplasy; mumps virus; perfect phylogeny; SARS-CoV-2; virus; West Nile virus; Yule-Harding model; Zika virus.].

Origin and Diversification of the Saguaro Cactus (Carnegiea gigantea): A Within-Species Phylogenomic Analysis.

Reconstructing accurate historical relationships within a species poses numerous challenges, not least in many plant groups in which gene flow is high enough to extend well beyond species boundaries. Nonetheless, the extent of tree-like history within a species is an empirical question on which it is now possible to bring large amounts of genome sequence to bear. We assess phylogenetic structure across the geographic range of the saguaro cactus, an emblematic member of Cactaceae, a clade known for extensive hybridization and porous species boundaries. Using 200 Gb of whole genome resequencing data from 20 individuals sampled from 10 localities, we assembled two data sets comprising 150,000 biallelic single nucleotide polymorphisms (SNPs) from protein coding sequences. From these, we inferred within-species trees and evaluated their significance and robustness using five qualitatively different inference methods. Despite the low sequence diversity, large census population sizes, and presence of wide-ranging pollen and seed dispersal agents, phylogenetic trees were well resolved and highly consistent across both data sets and all methods. We inferred that the most likely root, based on marginal likelihood comparisons, is to the east and south of the region of highest genetic diversity, which lies along the coast of the Gulf of California in Sonora, Mexico. Together with striking decreases in marginal likelihood found to the north, this supports hypotheses that saguaro's current range reflects postglacial expansion from the refugia in the south of its range. We conclude with observations about practical and theoretical issues raised by phylogenomic data sets within species, in which SNP-based methods must be used rather than gene tree methods that are widely used when sequence divergence is higher. These include computational scalability, inference of gene flow, and proper assessment of statistical support in the presence of linkage effects. [Phylogenomics; phylogeography; rooting; Sonoran Desert.].

The Implications of Lineage-Specific Rates for Divergence Time Estimation.

Rate variation adds considerable complexity to divergence time estimation in molecular phylogenies. Here, we evaluate the impact of lineage-specific rates-which we define as among-branch-rate-variation that acts consistently across the entire genome. We compare its impact to residual rates-defined as among-branch-rate-variation that shows a different pattern of rate variation at each sampled locus, and gene-specific rates-defined as variation in the average rate across all branches at each sampled locus. We show that lineage-specific rates lead to erroneous divergence time estimates, regardless of how many loci are sampled. Further, we show that stronger lineage-specific rates lead to increasing error. This contrasts to residual rates and gene-specific rates, where sampling more loci significantly reduces error. If divergence times are inferred in a Bayesian framework, we highlight that error caused by lineage-specific rates significantly reduces the probability that the 95% highest posterior density includes the correct value, and leads to sensitivity to the prior. Use of a more complex rate prior-which has recently been proposed to model rate variation more accurately-does not affect these conclusions. Finally, we show that the scale of lineage-specific rates used in our simulation experiments is comparable to that of an empirical data set for the angiosperm genus Ipomoea. Taken together, our findings demonstrate that lineage-specific rates cause error in divergence time estimates, and that this error is not overcome by analyzing genomic scale multilocus data sets. [Divergence time estimation; error; rate variation.].

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales.

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

On the dynamical structure of calcium oscillations.

Oscillations in the concentration of free cytosolic Ca2+ are an important and ubiquitous control mechanism in many cell types. It is thus correspondingly important to understand the mechanisms that underlie the control of these oscillations and how their period is determined. We show that Class I Ca2+ oscillations (i.e., oscillations that can occur at a constant concentration of inositol trisphosphate) have a common dynamical structure, irrespective of the oscillation period. This commonality allows the construction of a simple canonical model that incorporates this underlying dynamical behavior. Predictions from the model are tested, and confirmed, in three different cell types, with oscillation periods ranging over an order of magnitude. The model also predicts that Ca2+ oscillation period can be controlled by modulation of the rate of activation by Ca2+ of the inositol trisphosphate receptor. Preliminary experimental evidence consistent with this hypothesis is presented. Our canonical model has a structure similar to, but not identical to, the classic FitzHugh-Nagumo model. The characterization of variables by speed of evolution, as either fast or slow variables, changes over the course of a typical oscillation, leading to a model without globally defined fast and slow variables.

Extensive gene tree discordance and hemiplasy shaped the genomes of North American columnar cacti.

Few clades of plants have proven as difficult to classify as cacti. One explanation may be an unusually high level of convergent and parallel evolution (homoplasy). To evaluate support for this phylogenetic hypothesis at the molecular level, we sequenced the genomes of four cacti in the especially problematic tribe Pachycereeae, which contains most of the large columnar cacti of Mexico and adjacent areas, including the iconic saguaro cactus (Carnegiea gigantea) of the Sonoran Desert. We assembled a high-coverage draft genome for saguaro and lower coverage genomes for three other genera of tribe Pachycereeae (Pachycereus, Lophocereus, and Stenocereus) and a more distant outgroup cactus, Pereskia We used these to construct 4,436 orthologous gene alignments. Species tree inference consistently returned the same phylogeny, but gene tree discordance was high: 37% of gene trees having at least 90% bootstrap support conflicted with the species tree. Evidently, discordance is a product of long generation times and moderately large effective population sizes, leading to extensive incomplete lineage sorting (ILS). In the best supported gene trees, 58% of apparent homoplasy at amino sites in the species tree is due to gene tree-species tree discordance rather than parallel substitutions in the gene trees themselves, a phenomenon termed "hemiplasy." The high rate of genomic hemiplasy may contribute to apparent parallelisms in phenotypic traits, which could confound understanding of species relationships and character evolution in cacti.

Intercellular Ca(2+) waves: mechanisms and function.

Intercellular calcium (Ca(2+)) waves (ICWs) represent the propagation of increases in intracellular Ca(2+) through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca(2+) from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

BACKGROUND: Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. OBJECTIVE: We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). METHODS: Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3Zp3-Cre mice, which do not have a blood supply. RESULTS: Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. CONCLUSION: Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients.

The prevalence of terraced treescapes in analyses of phylogenetic data sets.

BACKGROUND: The pattern of data availability in a phylogenetic data set may lead to the formation of terraces, collections of equally optimal trees. Terraces can arise in tree space if trees are scored with parsimony or with partitioned, edge-unlinked maximum likelihood. Theory predicts that terraces can be large, but their prevalence in contemporary data sets has never been surveyed. We selected 26 data sets and phylogenetic trees reported in recent literature and investigated the terraces to which the trees would belong, under a common set of inference assumptions. We examined terrace size as a function of the sampling properties of the data sets, including taxon coverage density (the proportion of taxon-by-gene positions with any data present) and a measure of gene sampling "sufficiency". We evaluated each data set in relation to the theoretical minimum gene sampling depth needed to reduce terrace size to a single tree, and explored the impact of the terraces found in replicate trees in bootstrap methods. RESULTS: Terraces were identified in nearly all data sets with taxon coverage densities < 0.90. They were not found, however, in high-coverage-density (i.e., ≥ 0.94) transcriptomic and genomic data sets. The terraces could be very large, and size varied inversely with taxon coverage density and with gene sampling sufficiency. Few data sets achieved a theoretical minimum gene sampling depth needed to reduce terrace size to a single tree. Terraces found during bootstrap resampling reduced overall support. CONCLUSIONS: If certain inference assumptions apply, trees estimated from empirical data sets often belong to large terraces of equally optimal trees. Terrace size correlates to data set sampling properties. Data sets seldom include enough genes to reduce terrace size to one tree. When bootstrap replicate trees lie on a terrace, statistical support for phylogenetic hypotheses may be reduced. Although some of the published analyses surveyed were conducted with edge-linked inference models (which do not induce terraces), unlinked models have been used and advocated. The present study describes the potential impact of that inference assumption on phylogenetic inference in the context of the kinds of multigene data sets now widely assembled for large-scale tree construction.

The flagellar protein Enkurin is required for mouse sperm motility and for transport through the female reproductive tract.

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.

Toward a Self-Updating Platform for Estimating Rates of Speciation and Migration, Ages, and Relationships of Taxa.

Rapidly growing biological data-including molecular sequences and fossils-hold an unprecedented potential to reveal how evolutionary processes generate and maintain biodiversity. However, researchers often have to develop their own idiosyncratic workflows to integrate and analyze these data for reconstructing time-calibrated phylogenies. In addition, divergence times estimated under different methods and assumptions, and based on data of various quality and reliability, should not be combined without proper correction. Here we introduce a modular framework termed SUPERSMART (Self-Updating Platform for Estimating Rates of Speciation and Migration, Ages, and Relationships of Taxa), and provide a proof of concept for dealing with the moving targets of evolutionary and biogeographical research. This framework assembles comprehensive data sets of molecular and fossil data for any taxa and infers dated phylogenies using robust species tree methods, also allowing for the inclusion of genomic data produced through next-generation sequencing techniques. We exemplify the application of our method by presenting phylogenetic and dating analyses for the mammal order Primates and for the plant family Arecaceae (palms). We believe that this framework will provide a valuable tool for a wide range of hypothesis-driven research questions in systematics, biogeography, and evolution. SUPERSMART will also accelerate the inference of a "Dated Tree of Life" where all node ages are directly comparable. [Bayesian phylogenetics; data mining; divide-and-conquer methods; GenBank; multilocus multispecies coalescent; next-generation sequencing; palms; primates; tree calibration.].

Store-operated calcium entry is required for sustained contraction and Ca2+ oscillations of airway smooth muscle.

KEY POINTS: Airway hyper-responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (ASMCs). Agonist-induced Ca2+ oscillations underlie this contraction of ASMCs and the magnitude of this contraction is proportional to the Ca2+ oscillation frequency. Sustained contraction and Ca2+ oscillations require an influx of extracellular Ca2+ , although the mechanisms and pathways mediating this Ca2+ influx during agonist-induced ASMC contraction are not well defined. By inhibiting store-operated calcium entry (SOCE) or voltage-gated Ca2+ channels (VGCCs), we show that SOCE, rather than Ca2+ influx via VGCCs, provides the major Ca2+ entry pathway into ASMCs to sustain ASMCs contraction and Ca2+ oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper-responsiveness in asthma. ABSTRACT: Asthma is characterized by airway hyper-responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca2+ oscillations within airway smooth muscle cells (ASMCs). Sustained Ca2+ oscillations require a Ca2+ influx to replenish Ca2+ losses across the plasma membrane. Our previous studies implied store-operated calcium entry (SOCE) as the major pathway for this Ca2+ influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975A and GSK5498A) as well as L-type voltage-gated Ca2+ channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca2+ oscillations and SOCE-mediated Ca2+ influx in ASMCs within mouse precision-cut lung slices. We found that both GSK7975A and GSK5498A were able to fully relax methacholine-induced airway contraction by abolishing the Ca2+ oscillations, in a manner similar to that observed in zero extracellular Ca2+ ([Ca2+ ]e ). In addition, GSK7975A and GSK5498A inhibited increases in intracellular Ca2+ ([Ca2+ ]i ) in ASMCs with depleted Ca2+ -stores in response to increased [Ca2+ ]e , demonstrating a response consistent with the inhibition of SOCE. However, GSK7975A and GSK5498A did not reduce Ca2+ release via IP3 receptors stimulated with IP3 released from caged-IP3 . By contrast, nifedipine and nimodipine only partially reduced airway contraction, Ca2+ oscillation frequency and SOCE-mediated Ca2+ influx. These data suggest that SOCE is the major Ca2+ influx pathway for ASMCs with respect to sustaining agonist-induced airway contraction and the underlying Ca2+ oscillations. The mechanisms of SOCE may therefore form novel targets for new bronchodilators.

The relative contributions of store-operated and voltage-gated Ca2+ channels to the control of Ca2+ oscillations in airway smooth muscle.

KEY POINTS: Agonist-dependent oscillations in the concentration of free cytosolic calcium are a vital mechanism for the control of airway smooth muscle contraction and thus are a critical factor in airway hyper-responsiveness. Using a mathematical model, closely tied to experimental work, we show that the oscillations in membrane potential accompanying the calcium oscillations have no significant effect on the properties of the calcium oscillations. In addition, the model shows that calcium entry through store-operated calcium channels is critical for calcium oscillations, but calcium entry through voltage-gated channels has much less effect. The model predicts that voltage-gated channels are less important than store-operated channels in the control of airway smooth muscle tone. ABSTRACT: Airway smooth muscle contraction is typically the key mechanism underlying airway hyper-responsiveness, and the strength of muscle contraction is determined by the frequency of oscillations of intracellular calcium (Ca2+ ) concentration. In airway smooth muscle cells, these Ca2+ oscillations are caused by cyclic Ca2+ release from the sarcoplasmic reticulum, although Ca2+ influx via plasma membrane channels is also necessary to sustain the oscillations over longer times. To assess the relative contributions of store-operated and voltage-gated Ca2+ channels to this Ca2+ influx, we generated a comprehensive mathematical model, based on experimental Ca2+ measurements in mouse precision-cut lung slices, to simulate Ca2+ oscillations and changes in membrane potential. Agonist-induced Ca2+ oscillations are accompanied by oscillations in membrane potential, although the membrane potential oscillations are too small to generate large Ca2+ currents through voltage-gated Ca2+ channels, and thus have little effect on the Ca2+ oscillations. Ca2+ entry through voltage-gated channels only becomes important when the cell is depolarized (e.g. by a high external K+ concentration). As a result, agonist-induced Ca2+ oscillations are critically dependent on Ca2+ entry through store-operated channels but do not depend strongly on Ca2+ entry though voltage-gated channels.

Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling.

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine (n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 (n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS (n = 4-7, P = nonsignificant), it did inhibit calcium oscillations (n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists.

Human precision-cut lung slices (hPCLSs) provide a unique ex vivo model for translational research. However, the limited and unpredictable availability of human lung tissue greatly impedes their use. Here, we demonstrate that cryopreservation of hPCLSs facilitates banking of live human lung tissue for routine use. Our results show that cryopreservation had little effect on overall cell viability and vital functions of immune cells, including phagocytes and T lymphocytes. In addition, airway contraction and relaxation in response to specific agonists and antagonists, respectively, were unchanged after cryopreservation. At the subcellular level, cryopreserved hPCLSs maintained Ca(2+)-dependent regulatory mechanisms for the control of airway smooth muscle cell contractility. To exemplify the use of cryopreserved hPCLSs in smooth muscle research, we provide evidence that bitter-taste receptor (TAS2R) agonists relax airways by blocking Ca(2+) oscillations in airway smooth muscle cells. In conclusion, the banking of cryopreserved hPCLSs provides a robust bioassay for translational research of lung physiology and disease.

Impacts of Terraces on Phylogenetic Inference.

Terraces are sets of trees with precisely the same likelihood or parsimony score, which can be induced by missing sequences in partitioned multi-locus phylogenetic data matrices. The potentially large set of trees on a terrace can be characterized by enumeration algorithms or consensus methods that exploit the pattern of partial taxon coverage in the data, independent of the sequence data themselves. Terraces can add ambiguity and complexity to phylogenetic inference, particularly in settings where inference is already challenging: data sets with many taxa and relatively few loci. In this article we present five new findings about terraces and their impacts on phylogenetic inference. First, we clarify assumptions about partitioning scheme model parameters that are necessary for the existence of terraces. Second, we explore the dependence of terrace size on partitioning scheme and indicate how to find the partitioning scheme associated with the largest terrace containing a given tree. Third, we highlight the impact of terrace size on bootstrap estimates of confidence limits in clades, and characterize the surprising result that the bootstrap proportion for a clade, as it is usually calculated, can be entirely determined by the frequency of bipartitions on a terrace, with some bipartitions receiving high support even when incorrect. Fourth, we dissect some effects of prior distributions of edge lengths on the computed posterior probabilities of clades on terraces, to understand an example in which long edges "attract" each other in Bayesian inference. Fifth, we describe how assuming relationships between edge-lengths of different loci, as an attempt to avoid terraces, can also be problematic when taxon coverage is partial, specifically when heterotachy is present. Finally, we discuss strategies for remediation of some of these problems. One promising approach finds a minimal set of taxa which, when deleted from the data matrix, reduces the size of a terrace to a single tree.

Ryanodine receptor sensitization results in abnormal calcium signaling in airway smooth muscle cells.

Intracellular Ca(2+) dynamics of airway smooth muscle cells (ASMCs) are believed to play a major role in airway hyperresponsiveness and remodeling in asthma. Prior studies have underscored a prominent role for inositol 1,4,5-triphosphate (IP3) receptors in normal agonist-induced Ca(2+) oscillations, whereas ryanodine receptors (RyRs) appear to remain closed during such Ca(2+) oscillations, which mediate ASMC contraction. Nevertheless, RyRs have been hypothesized to play a role in hyperresponsive Ca(2+) signaling. This could be explained by RyRs being "sensitized" to open more frequently by certain compounds. We investigate the implications of RyR sensitization on Ca(2+) dynamics in ASMC using a combination of mathematical modeling and experiments with mouse precision-cut lung slices. Caffeine is used to increase the sensitivity of RyRs to cytosolic Ca(2+) concentration ([Ca(2+)]i) and sarcoplasmic reticulum Ca(2+) ([Ca(2+)]SR). In ASMCs, high caffeine concentrations (>10 mM) induce a sustained elevation of [Ca(2+)]i. Our mathematical model accounts for this by the activation of store-operated Ca(2+) entry that results from a large increase in the RyR sensitivity to [Ca(2+)]SR and the associated Ca(2+) release, which leads to a reduction of [Ca(2+)]SR. Importantly, our model also predicts that: (1) moderate RyR sensitization induces slow Ca(2+) oscillations, a result experimentally confirmed with low concentrations of caffeine; and (2) high RyR sensitization suppresses fast, agonist-induced Ca(2+) oscillations by inducing substantial store-operated Ca(2+) entry and elevated [Ca(2+)]i. These results suggest that RyR sensitization could play a role in ASMC proliferation (by inducing slow Ca(2+) oscillations) and in airway hyperresponsiveness (by inducing greater mean [Ca(2+)]i for similar levels of contractile agonist).

Ceiba: scalable visualization of phylogenies and 2D/3D image collections.

UNLABELLED: Phylogenetic trees with hundreds of thousands of leaves are now being inferred from sequence data, posing significant challenges for visualization and exploratory analysis. Image data supplying valuable context for species in trees (and cues for exploring them) are becoming increasingly available in biodiversity databases and elsewhere but have rarely been built into tree visualization software in a scalable way. Ceiba lets the user explore large trees and inspect image collection arrays (sets of 'homologous' images) comprising mixtures of 2D and 3D image objects. Ceiba exploits recent improvements in graphics hardware, OpenGL toolkits and many standard high-performance computer graphics strategies, such as texture compression, level of detail control, culling, animations and image caching. Its tree layouts can be tuned by user-provided phylogenetic definitions of subtrees. The code has been extensively tested on phylogenies of up to 55,000 leaves and images. AVAILABILITY AND IMPLEMENTATION: A manual, datasets, source code (distributed under GPL) and binaries for OS X are available at http://sourceforge.net/projects/ceiba. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pulmonary vein sleeve cell excitation-contraction-coupling becomes dysynchronized by spontaneous calcium transients.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia. Substantial evidence indicates that cardiomyocytes located in the pulmonary veins [pulmonary vein sleeve cells (PVCs)] cause AF by generating ectopic electrical activity. Electrical ablation, isolating PVCs from their left atrial junctions, is a major treatment for AF. In small rodents, the sleeve of PVCs extends deep inside the lungs and is present in lung slices. Here we present data, using the lung slice preparation, characterizing how spontaneous Ca2+ transients in PVCs affect their capability to respond to electrical pacing. Immediately after a spontaneous Ca2+ transient the cell is in a refractory period and it cannot respond to electrical stimulation. Consequently, we observe that the higher the level of spontaneous activity in an individual PVC, the less likely it is that this PVC responds to electrical field stimulation. The spontaneous activity of neighbouring PVCs can be different from each other. Heterogeneity in the Ca2+ signalling of cells and in their responsiveness to electrical stimuli are known pro-arrhythmic events. The tendency of PVCs to show spontaneous Ca2+ transients and spontaneous action potentials (APs) underlies their potential to cause AF.

Confluence, synnovation, and depauperons in plant diversification.

We review the empirical phylogenetic literature on plant diversification, highlighting challenges in separating the effects of speciation and extinction, in specifying diversification mechanisms, and in making convincing arguments. In recent discussions of context dependence, key opportunities and landscapes, and indirect effects and lag times, we see a distinct shift away from single-point/single-cause 'key innovation' hypotheses toward more nuanced explanations involving multiple interacting causal agents assembled step-wise through a tree. To help crystalize this emerging perspective we introduce the term 'synnovation' (a hybrid of 'synergy' and 'innovation') for an interacting combination of traits with a particular consequence ('key synnovation' in the case of increased diversification rate), and the term 'confluence' for the sequential coming together of a set of traits (innovations and synnovations), environmental changes, and geographic movements along the branches of a phylogenetic tree. We illustrate these concepts using the radiation of Bromeliaceae. We also highlight the generality of these ideas by considering how rate heterogeneity associated with a confluence relates to the existence of particularly species-poor lineages, or 'depauperons.' Many challenges are posed by this re-purposed research framework, including difficulties associated with partial taxon sampling, uncertainty in divergence time estimation, and extinction.