A phylogenetic method for detecting positive epistasis in gene sequences and its application to RNA virus evolution

RNA virus genomes are compact, often containing multiple overlapping reading frames and functional secondary structure. Consequently, it is thought that evolutionary interactions between nucleotide sites are commonplace in the genomes of these infectious agents. However, the role of epistasis in natural populations of RNA viruses remains unclear. To investigate the pervasiveness of epistasis in RNA viruses, we used a parsimony-based computational method to identify pairs of co-occurring mutations along phylogenies of 177 RNA virus genes. This analysis revealed widespread evidence for positive epistatic interactions at both synonymous and nonsynonymous nucleotide sites and in both clonal and recombining viruses, with the majority of these interactions spanning very short sequence regions. These findings have important implications for understanding the key aspects of RNA virus evolution, including the dynamics of adaptation. Additionally, many comparative analyses that utilize the phylogenetic relationships among gene sequences assume that mutations represent independent, uncorrelated events. Our results show that this assumption may often be invalid.     

Theory of the mitotic index and its application to tissue growth measurement

Analysis is made to show that the mitotic index is simply proportional to the ratio of the duration of mitosis (T) to the intermitotic time only under special conditions. In the case of exponential growth of cell population the simple proportionality hold if the product ofT and the growth constant is small. For power law (t ⁿ ) growth of cell population the simple proportionality holds only when a steady state of growth has existed for at least ten intermitotic periods. The simple proportionality does not apply in conditions of transient growth. This document is based on work performed at Los Alamos Scientific Laboratory of the University of California under government contract W-7405-eng-36 and the information contained therein will appear in Division V of the National Nuclear Energy Series (Manhattan Project Technical Section) as part of the contributions of the Los Alamos Laboratory.     

Prevalence of epistasis in the evolution of influenza A surface proteins

The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with , which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.     

An immunocytochemical method for histamine: application to the planarians

Histamine-immunoreactivity was investigated in the planarians Dugesia tigrina and Polycelis nigra. Specific antisera against a histamine-protein conjugate were used, and 1-ethyl—3 (3-dimethyl-aminopropyl) carbodiimide was used both as coupling agent to prepare the antigen and as a tissue fixative. In D. tigrina, histamine-immunoreactivity was restricted to photoreceptor cells in the cerebral eye. In P. nigra, nerve fibers were found in the ventral nerve cord and nerves running laterally from these. The epidermal eyes did not display histamine-immunoreactivity. The results suggest that histamine may be a transmitter in some of the most primitive animals. They also suggest that the distribution of histamine may differ in planarians.     

An end to endless forms: epistasis, phenotype distribution bias, and nonuniform evolution

Studies of the evolution of development characterize the way in which gene regulatory dynamics during ontogeny constructs and channels phenotypic variation. These studies have identified a number of evolutionary regularities: (1) phenotypes occupy only a small subspace of possible phenotypes, (2) the influence of mutation is not uniform and is often canalized, and (3) a great deal of morphological variation evolved early in the history of multicellular life. An important implication of these studies is that diversity is largely the outcome of the evolution of gene regulation rather than the emergence of new, structural genes. Using a simple model that considers a generic property of developmental maps-the interaction between multiple genetic elements and the nonlinearity of gene interaction in shaping phenotypic traits-we are able to recover many of these empirical regularities. We show that visible phenotypes represent only a small fraction of possibilities. Epistasis ensures that phenotypes are highly clustered in morphospace and that the most frequent phenotypes are the most similar. We perform phylogenetic analyses on an evolving, developmental model and find that species become more alike through time, whereas higher-level grades have a tendency to diverge. Ancestral phenotypes, produced by early developmental programs with a low level of gene interaction, are found to span a significantly greater volume of the total phenotypic space than derived taxa. We suggest that early and late evolution have a different character that we classify into micro- and macroevolutionary configurations. These findings complement the view of development as a key component in the production of endless forms and highlight the crucial role of development in constraining biotic diversity and evolutionary trajectories.     

The geometry of shape space: application to influenza

Shape space was proposed over 20 years ago as a conceptual formalism in which to represent antibody/antigen binding. It has since played a key role in computational immunology. Antigens and antibodies are considered to be points in an abstract "shape space", where coordinates of points in this space represent generalized physico-chemical properties associated with various (unspecified) physical properties related to binding, such as geometric shape, hydrophobicity, charge, etc. Distances in shape space between points representing antibodies and (the shape complement) of antigens are assumed to be related to their affinity, with small distances corresponding to high affinity. In this paper, we provide algorithms, related to metric and ordinal multidimensional scaling algorithms first developed in the mathematical psychology literature, which construct explicit, quantitative coordinates for points in shape space given experimental data such as hemagglutination inhibition assays, or other general affinity assays. Previously, such coordinates had been conceptual constructs and totally implicit. The dimension of shape space deduced from hemagglutination inhibition assays for influenza is low, approximately five dimensional.The deduction of the explicit geometry of shape space given experimental affinity data provides new ways to quantify the similarity of antibodies to antibodies, antigens to antigens, and the affinity of antigens to antibodies. This has potential utility in, e.g. strain selection decisions for annual influenza vaccines, among other applications. The analysis techniques presented here are not restricted to the analysis of antibody-antigen interactions and are generally applicable to affinity data resulting from binding assays.     

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)–TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006–2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.     

Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.     

An ELISA test for BA measurement: application to wild cherry culture in vitro

An avidin-biotin solid phase enzyme linked immunoassay (ELISA) was developed for benzyladenine (BA) measurement in HPLC purified extracts. Its measuring range extended from 10 fmol to 10 pmol. Analysis of the immunoreactivity of all fractions obtained by HPLC fractionation of a methanolic extract from in vitro cultured wild cherry shoots showed that BA was accumulated in plantlets with 2 other immunoreactive compounds. Interassay variability never exceeded 3%.     

Bisubstrate inhibition: Theory and application to N-acetyltransferases

Bisubstrate inhibitors represent a potentially powerful group of compounds that have found significant therapeutic utility. Although these compounds have been synthesized and tested against a number of enzymes that catalyze sequential bireactant reactions, the detailed theory for predicting the expected patterns of inhibition against the two substrates for various bireactant kinetic mechanisms has, heretofore, not been presented. We have derived the rate equations for all likely sequential bireactant mechanisms and provide two examples in which bisubstrate inhibitors allow the kinetic mechanism to be determined. Bisubstrate inhibitor kinetics is a powerful diagnostic for the determination of kinetic mechanisms.     

An image-based method to automatically propagate bony landmarks: application to computational spine biomechanics

In attempts to elucidate the underlying mechanisms of spinal injuries and spinal deformities, several experimental and numerical studies have been conducted to understand the biomechanical behavior of the spine. However, numerical biomechanical studies suffer from uncertainties associated with hard- and soft-tissue anatomies. Currently, these parameters are identified manually on each mesh model prior to simulations. The determination of soft connective tissues on finite element meshes can be a tedious procedure, which limits the number of models used in the numerical studies to a few instances. In order to address these limitations, an image-based method for automatic morphing of soft connective tissues has been proposed. Results showed that the proposed method is capable to accurately determine the spatial locations of predetermined bony landmarks. The present method can be used to automatically generate patient-specific models, which may be helpful in designing studies involving a large number of instances and to understand the mechanical behavior of biomechanical structures across a given population.     

Cuckoo search epistasis: a new method for exploring significant genetic interactions

The advent of high-throughput sequencing technology has resulted in the ability to measure millions of single-nucleotide polymorphisms (SNPs) from thousands of individuals. Although these high-dimensional data have paved the way for better understanding of the genetic architecture of common diseases, they have also given rise to challenges in developing computational methods for learning epistatic relationships among genetic markers. We propose a new method, named cuckoo search epistasis (CSE) for identifying significant epistatic interactions in population-based association studies with a case–control design. This method combines a computationally efficient Bayesian scoring function with an evolutionary-based heuristic search algorithm, and can be efficiently applied to high-dimensional genome-wide SNP data. The experimental results from synthetic data sets show that CSE outperforms existing methods including multifactorial dimensionality reduction and Bayesian epistasis association mapping. In addition, on a real genome-wide data set related to Alzheimer''s disease, CSE identified SNPs that are consistent with previously reported results, and show the utility of CSE for application to genome-wide data.     

Quantitative method for measurement of aerotolerance of bacteria and its application to oral indigenous anaerobes

An index which expressed the aerobic to anaerobic potential was made for bacteria with intermediate tolerance for oxygen. One method used for this analysis was measurement of the relative bacterial growth ratio. The other method was based on the pattern of the absorbancy versus depth plot. The index was applied to oral indigenous anaerobes.     

Contributions of heterosis and epistasis to hybrid fitness

Early-generation hybrid fitness is difficult to interpret because heterosis can obscure the effects of hybrid breakdown. We used controlled reciprocal crosses and common garden experiments to distinguish between effects of heterosis and nuclear and cytonuclear epistasis among morphotypes and advanced-generation hybrid derivative populations in the Piriqueta caroliniana (Turneraceae) plant complex. Seed germination, growth, and sexual reproduction of first-generation hybrids, inbred parental lines, and outbred parental lines were compared under field conditions. Average vegetative performance was greater for hybrids than for inbred lines, and first-season growth was similar for hybrids and outbred parental lines. Hybrid survival surpassed that of inbred lines and was equal to or greater than outbred lines' survival, and more F(1) than parental plants reproduced. Reductions in hybrid fitness due to Dobzhansky-Muller incompatibilities (epistasis among divergent genetic elements) were expressed as differences in vegetative growth, survival, and reproduction between plants from reciprocal crosses for both F(1) and backcross hybrid generations. Comparing performance of hybrids against parental genotypes from intra- and interpopulation crosses allowed a more robust prediction of F(1) hybrids' success and more accurate interpretations of the genetic architecture of F(1) hybrid vigor.     

Biodiversity as insurance: from concept to measurement and application

Biological insurance theory predicts that, in a variable environment, aggregate ecosystem properties will vary less in more diverse communities because declines in the performance or abundance of some species or phenotypes will be offset, at least partly, by smoother declines or increases in others. During the past two decades, ecology has accumulated strong evidence for the stabilising effect of biodiversity on ecosystem functioning. As biological insurance is reaching the stage of a mature theory, it is critical to revisit and clarify its conceptual foundations to guide future developments, applications and measurements. In this review, we first clarify the connections between the insurance and portfolio concepts that have been used in ecology and the economic concepts that inspired them. Doing so points to gaps and mismatches between ecology and economics that could be filled profitably by new theoretical developments and new management applications. Second, we discuss some fundamental issues in biological insurance theory that have remained unnoticed so far and that emerge from some of its recent applications. In particular, we draw a clear distinction between the two effects embedded in biological insurance theory, i.e. the effects of biodiversity on the mean and variability of ecosystem properties. This distinction allows explicit consideration of trade-offs between the mean and stability of ecosystem processes and services. We also review applications of biological insurance theory in ecosystem management. Finally, we provide a synthetic conceptual framework that unifies the various approaches across disciplines, and we suggest new ways in which biological insurance theory could be extended to address new issues in ecology and ecosystem management. Exciting future challenges include linking the effects of biodiversity on ecosystem functioning and stability, incorporating multiple functions and feedbacks, developing new approaches to partition biodiversity effects across scales, extending biological insurance theory to complex interaction networks, and developing new applications to biodiversity and ecosystem management.     

An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored.