Isolation, characterisation and molecular evolution of the actin gene family of the New Zealand black-footed abalone, Haliotis iris.

Molluscs are biologically important; they form a diverse taxon and the most well known members of the Lophotrochozoa, the most understudied Bilaterian group. Despite this importance, few studies have characterised molluscs at the genetic level. The New Zealand black-footed abalone Haliotis iris, an economically and culturally valued species, was chosen as a model for genetic characterisation of a molluscan actin gene family. In H. iris, actin is essential for the production of a large muscular foot, which forms the bulk of the body mass. The structure, expression and evolution of actin genes were investigated to elucidate the function of the actin gene family in H. iris. H. iris actin genes were isolated by PCR using gene subtype-specific primers designed from previously characterised partial H. iris actin sequences and generic primers derived from H. rufescens (Californian red abalone) and Cyprinus carpio (common carp). Three full length genes, H.irisA1, H.irisA2 and H.irisA3, and three partial genes, H.irisA1a, H.irisA1b and H.irisA1c, were isolated. The full length genes showed 82-95% sequence similarity to mollusc actin gene sequences deposited in GenBank. Sequence conservation confirmed the identity of the putative actin genes. The six genes contained a single variable length intron between codons 41 and 42. Intron lengths were: 174 nt, H.irisA1; 1,078 nt, H.irisA2; 581 nt, H.irisA3; 301 nt, H.irisA1a; 282 nt, H.iris1l and 229 nt, H.irisA1c. The predicted proteins of the full length genes contained 375 aa and lacked the second amino acid usually found in invertebrate actin proteins. Southern hybridisation of genomic DNA suggested there was a large gene family composed of at least eight members. The expression of H.irisA1, H.irisA2 and H.irisA3 in developmental stages and adult tissues was investigated by RT-PCR. RT-PCR demonstrated differential expression of H. iris actin genes during development and in adult tissues. H.irisA1 and H.irisA2 were expressed at low levels in fertilised eggs and blastula, with expression increasing in trochophore and veliger larvae. H.irisA3 was not expressed in eggs, but was faintly detected in blastula and highly expressed in trochophore and veliger larvae. H.irisA1 was ubiquitously expressed in adult gill, gonad, hepatopancreas, foot and mantle tissue, suggesting it may be a cytoplasmic-type actin. H.irisA2 was expressed in all tissues except the hepatopancreas, although low expression may not have been detectable by electrophoresis of RT -PCR products. Further characterisation is required to confirm whether H.irisA2 encodes a cytoplasmic-type actin. H.irisA3 was expressed at high levels in the muscular foot and mantle, and was faintly detected in gonad, suggesting it may be a muscle type actin. Phylogenetic analyses of H. iris actin genes and other molluscan actin genes available on GenBank were performed using maximum parsimony and maximum likelihood methods. Analyses suggested that haliotid actins can be divided into two orthologous clades, the first clade containing H.irisA1, H.irisA1a, H.irisA1b, H.irisA1c, H.virgA1a, H.virgA1b, H.virgA1c and H. Rufescens actin, the second clade containing H.irisA2, H.irisA3 and H. discus hannai actin. Orthology indicated that the last common ancestor of haliotids had at least two actin genes. Clustering of actin genes from individual haliotid species within orthologous actin gene clades suggests paralogy resulting from duplication of actin genes within species. Evidence for gene orthology between mollusc actin genes was found, but further characterisation of actin genes from other mollusc species is required to infer the evolutionary significance of orthology

Cell-specific responses to the cytokine TGFβ are determined by variability in protein levels

The cytokine TGFβ provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFβ are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time-resolved measurements of pathway activation at the single-cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single-cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock-out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity

Bistability in Apoptosis by Receptor Clustering

Apoptosis is a highly regulated cell death mechanism involved in many physiological processes. A key component of extrinsically activated apoptosis is the death receptor Fas, which, on binding to its cognate ligand FasL, oligomerize to form the death-inducing signaling complex. Motivated by recent experimental data, we propose a mathematical model of death ligand-receptor dynamics where FasL acts as a clustering agent for Fas, which form locally stable signaling platforms through proximity-induced receptor interactions. Significantly, the model exhibits hysteresis, providing an upstream mechanism for bistability and robustness. At low receptor concentrations, the bistability is contingent on the trimerism of FasL. Moreover, irreversible bistability, representing a committed cell death decision, emerges at high concentrations, which may be achieved through receptor pre-association or localization onto membrane lipid rafts. Thus, our model provides a novel theory for these observed biological phenomena within the unified context of bistability. Importantly, as Fas interactions initiate the extrinsic apoptotic pathway, our model also suggests a mechanism by which cells may function as bistable life/death switches independently of any such dynamics in their downstream components. Our results highlight the role of death receptors in deciding cell fate and add to the signal processing capabilities attributed to receptor clustering.Comment: Accepted by PLoS Comput Bio

Symbolic Versus Numerical Computation and Visualization of Parameter Regions for Multistationarity of Biological Networks

We investigate models of the mitogenactivated protein kinases (MAPK) network, with the aim of determining where in parameter space there exist multiple positive steady states. We build on recent progress which combines various symbolic computation methods for mixed systems of equalities and inequalities. We demonstrate that those techniques benefit tremendously from a newly implemented graph theoretical symbolic preprocessing method. We compare computation times and quality of results of numerical continuation methods with our symbolic approach before and after the application of our preprocessing.Comment: Accepted into Proc. CASC 201

Employing the Metabolic “Branch Point Effect” to Generate an All-or-None, Digital-like Response in Enzymatic Outputs and Enzyme-Based Sensors

Here, we demonstrate a strategy to convert the graded Michaelis−Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the “branch point effect”, a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter’s response. Above an arbitrarily selected “threshold” substrate concentration, the nonsignaling HK enzyme saturates leading to a “sudden” activation of the first signaling GOx enzyme and a far steeper dose−response curve than that observed for simple Michaelis−Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

Bcl-2 inhibits apoptosis by increasing the time-to-death and intrinsic cell-to-cell variations in the mitochondrial pathway of cell death

BH3 mimetics have been proposed as new anticancer therapeutics. They target anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in the resistance of many cancer cells, particularly leukemia and lymphoma cells, to apoptosis. Using probabilistic computational modeling of the mitochondrial pathway of apoptosis, verified by single-cell experimental observations, we develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2 imparts its anti-apoptotic role by increasing the time-to-death and cell-to-cell variability. We also show that although the commitment to death is highly impacted by differences in protein levels at the time of stimulation, inherent stochastic fluctuations in apoptotic signaling are sufficient to induce cell-to-cell variability and to allow single cells to escape death. This study suggests that intrinsic cell-to-cell stochastic variability in apoptotic signaling is sufficient to cause fractional killing of cancer cells after exposure to BH3 mimetics. This is an unanticipated facet of cancer chemoresistance.Comment: 11 pages, In pres

Methodological developments in violence research

Über Jahrzehnte wurde Gewalt durch Interviews mit Betroffenen oder Tätern, durch teilnehmende Beobachtung oder Gewaltstatistiken untersucht, meist unter Verwendung entweder qualitativer oder quantitativer Analysemethoden. Seit der Jahrhundertwende stehen Forschenden eine Reihe neuer Ansätze zur Verfügung: Es gibt immer mehr Videoaufnahmen von gewaltsamen Ereignissen, Mixed Methods-Ansätze werden stetig weiterentwickelt und durch Computational Social Sciences finden Big Data-Ansätze Einzug in immer mehr Forschungsfelder. Diese drei Entwicklungen bieten großes Potenzial für die quantitative und qualitative Gewaltforschung. Der vorliegende Beitrag diskutiert Videodatenanalyse, Triangulation und Mixed Methods-Ansätze sowie Big Data und bespricht den gegenwärtigen und zukünftigen Einfluss der genannten Entwicklungen auf das Forschungsfeld. Das Augenmerk liegt besonders darauf, (1) wie neuere Videodaten genutzt werden können, um Gewalt zu untersuchen und wo ihre Vor- und Nachteile liegen, (2) wie Triangulation und Mixed Methods-Ansätze umfassendere Analysen und theoretische Verknüpfungen in der Gewaltforschung ermöglichen und (3) wo Anwendungen von Big Data und Computational Social Science in der Gewaltforschung liegen können.For decades violence research has relied on interviews with victims and perpetrators, on participant observation, and on survey methods, and most studies focused on either qualitative or quantitative analytic strategies. Since the turn of the millennium, researchers can draw on a range of new approaches: there are increasing amounts of video data of violent incidents, triangulation and mixed methods approaches become ever more sophisticated, and computational social sciences introduce big data analysis to more and more research fields. These three developments hold great potential for quantitative and qualitative violence research. This paper discusses video data analysis, mixed methods, and big data in the context of current and future violence research. Specific focus lies on (1) potentials and challenges of new video data for studying violence; (2) the role of triangulation and mixed methods in enabling more comprehensive violence research from multiple theoretical perspectives, and (3) what potential uses of big data and computational social science in violence research may look like

Sophisticated Framework between Cell Cycle Arrest and Apoptosis Induction Based on p53 Dynamics

The tumor suppressor, p53, regulates several gene expressions that are related to the DNA repair protein, cell cycle arrest and apoptosis induction, which activates the implementation of both cell cycle arrest and induction of apoptosis. However, it is not clear how p53 specifically regulates the implementation of these functions. By applying several well-known kinetic mathematical models, we constructed a novel model that described the influence that DNA damage has on the implementation of both the G2/M phase cell cycle arrest and the intrinsic apoptosis induction via its activation of the p53 synthesis process. The model, which consisted of 32 dependent variables and 115 kinetic parameters, was used to examine interference by DNA damage in the implementation of both G2/M phase cell cycle arrest and intrinsic apoptosis induction. A low DNA damage promoted slightly the synthesis of p53, which showed a sigmoidal behavior with time. In contrast, in the case of a high DNA damage, the p53 showed an oscillation behavior with time. Regardless of the DNA damage level, there were delays in the G2/M progression. The intrinsic apoptosis was only induced in situations where grave DNA damage produced an oscillation of p53. In addition, to wreck the equilibrium between Bcl-2 and Bax the induction of apoptosis required an extreme activation of p53 produced by the oscillation dynamics, and was only implemented after the release of the G2/M phase arrest. When the p53 oscillation is observed, there is possibility that the cell implements the apoptosis induction. Moreover, in contrast to the cell cycle arrest system, the apoptosis induction system is responsible for safeguarding the system that suppresses malignant transformations. The results of these experiments will be useful in the future for elucidating of the dominant factors that determine the cell fate such as normal cell cycles, cell cycle arrest and apoptosis

Bistability and Oscillations in Gene Regulation Mediated by Small Noncoding RNAs

The interplay of small noncoding RNAs (sRNAs), mRNAs, and proteins has been shown to play crucial roles in almost all cellular processes. As key post-transcriptional regulators of gene expression, the mechanisms and roles of sRNAs in various cellular processes still need to be fully understood. When participating in cellular processes, sRNAs mainly mediate mRNA degradation or translational repression. Here, we show how the dynamics of two minimal architectures is drastically affected by these two mechanisms. A comparison is also given to reveal the implication of the fundamental differences. This study may help us to analyze complex networks assembled by simple modules more easily. A better knowledge of the sRNA-mediated motifs is also of interest for bio-engineering and artificial control