Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Evolution of adaptive diversity and genetic connectivity in Arctic charr (Salvelinus alpinus) in Iceland

The ecological theory of adaptive radiation predicts that the evolution of phenotypic diversity within species is generated by divergent natural selection arising from different environments and competition between species. Genetic connectivity among populations is likely also to have an important role in both the origin and maintenance of adaptive genetic diversity. Our goal was to evaluate the potential roles of genetic connectivity and natural selection in the maintenance of adaptive phenotypic differences among morphs of Arctic charr, Salvelinus alpinus, in Iceland. At a large spatial scale, we tested the predictive power of geographic structure and phenotypic variation for patterns of neutral genetic variation among populations throughout Iceland. At a smaller scale, we evaluated the genetic differentiation between two morphs in Lake Thingvallavatn relative to historically explicit, coalescent-based null models of the evolutionary history of these lineages. At the large spatial scale, populations are highly differentiated, but weakly structured, both geographically and with respect to patterns of phenotypic variation. At the intralacustrine scale, we observe modest genetic differentiation between two morphs, but this level of differentiation is nonetheless consistent with strong reproductive isolation throughout the Holocene. Rather than a result of the homogenizing effect of gene flow in a system at migration-drift equilibrium, the modest level of genetic differentiation could equally be a result of slow neutral divergence by drift in large populations. We conclude that contemporary and recent patterns of restricted gene flow have been highly conducive to the evolution and maintenance of adaptive genetic variation in Icelandic Arctic charr.     

The Pseudomonas quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour

The Pseudomonas quinolone signal (PQS), and its precursor 2-heptyl-4-quinolone (HHQ), play a key role in coordinating virulence in the important cystic fibrosis pathogen Pseudomonas aeruginosa. The discovery of HHQ analogues in Burkholderia and other microorganisms led us to investigate the possibility that these compounds can influence interspecies behaviour. We found that surface-associated phenotypes were repressed in Gram-positive and Gram-negative bacteria as well as in pathogenic yeast in response to PQS and HHQ. Motility was repressed in a broad range of bacteria, while biofilm formation in Bacillus subtilis and Candida albicans was repressed in the presence of HHQ, though initial adhesion was unaffected. Furthermore, HHQ exhibited potent bacteriostatic activity against several Gram-negative bacteria, including pathogenic Vibrio vulnificus. Structure-function analysis using synthetic analogues provided an insight into the molecular properties that underpin the ability of these compounds to influence microbial behaviour, revealing the alkyl chain to be fundamental. Defining the influence of these molecules on microbial-eukaryotic-host interactions will facilitate future therapeutic strategies which seek to combat microorganisms that are recalcitrant to conventional antimicrobial agents.     

Changes in spatial and temporal distribution and vocal behavior of Blainville's beaked whales (Mesoplodon densirostris) during multiship exercises with mid‐frequency sonar

The number and distribution of vocalizing groups of Blainville's beaked whales (Mesoplodon densirostris) were analyzed before, during, and after multiship mid‐frequency active sonar operations at the US Navy's Atlantic Undersea Test and Evaluation Center (AUTEC) in the Bahamas. Groups of foraging animals were isolated by detecting their echolocation clicks using an array of bottom‐mounted hydrophones. Two data sets were evaluated consisting of 115 and 240 h of acoustic data in May 2007 and 2008, respectively. Vocal activity was observed to decline during active sonar exercises and increase upon cessation of sonar transmissions in both data sets. Vocal activity did not recover to preexposure levels in the postexposure time period in 2007 nor in the initial postexposure period in the 2008 data set. Clicks detected during sonar operations were generally found to be on the periphery of the hydrophone field and vocal durations declined for those groups that remained on the range in that time period. Receive levels were calculated for several vocal groups of whales and indicated that animals continued to forage when exposed to sonar at levels as high as 157 dB re: μPa.     

Inferring the time-invariant topology of a nonlinear sparse gene regulatory network using fully Bayesian spline autoregression

We propose a semiparametric Bayesian model, based on penalized splines, for the recovery of the time-invariant topology of a causal interaction network from longitudinal data. Our motivation is inference of gene regulatory networks from low-resolution microarray time series, where existence of nonlinear interactions is well known. Parenthood relations are mapped by augmenting the model with kinship indicators and providing these with either an overall or gene-wise hierarchical structure. Appropriate specification of the prior is crucial to control the flexibility of the splines, especially under circumstances of scarce data; thus, we provide an informative, proper prior. Substantive improvement in network inference over a linear model is demonstrated using synthetic data drawn from ordinary differential equation models and gene expression from an experimental data set of the Arabidopsis thaliana circadian rhythm.     

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.     

Sphingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana

Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.     

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.     

Into the wild—WAMBAM goes to Canada

The sixth Wild Animal Models Bi‐Annual Meeting was held in July 2017 in Québec, with 42 participants. This report documents the evolution of questions asked and approaches used in evolutionary quantitative genetic studies of wild populations in recent decades, and how these questions and approaches were represented at the recent meeting. We explore how ideas from previous meetings in this series have developed to their present states, and consider how the format of the meetings may be particularly useful at fostering the rapid development and proliferation of ideas and approaches.