Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.     

Multi-Platform Metabolomic Analyses of Ergosterol-Induced Dynamic Changes in Nicotiana tabacum Cells

Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0–1000 nM) of ergosterol and incubated for different time periods (0–24 h). Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC×GC-TOF-MS, UHPLC-MS) and ¹H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The ‘defensome’ involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin) and other metabolites (abscisic acid and phytosterols) putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.     

Fodis: Software for Protein Unfolding Analysis

The folding dynamics of proteins at the single-molecule level has been studied with single-molecule force spectroscopy experiments for 20 years, but a common standardized method for the analysis of the collected data and for sharing among the scientific community members is still not available. We have developed a new open-source tool—Fodis—for the analysis of the force-distance curves obtained in single-molecule force spectroscopy experiments, providing almost automatic processing, analysis, and classification of the obtained data. Our method provides also a classification of the possible unfolding pathways and the structural heterogeneity present during the unfolding of proteins.     

Compatible solutes as protectants for zymogens against proteolysis

Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800 mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4 h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.     

Physical Analysis of Tissue Mechanics in Amphibian Gastrulation

During morphogenesis, intercellular attachments constrain cellmobility so that embryonic tissues may (i) deform as solid sheetsof tightly bound cells, (ii) disperse to migrate as separatecells or (iii) flow as multicellular liquids (in which cellsremain aggregated yet can still slide past one another). Bymodelling deep germ layers as multicellular liquids in amphibiangastrulation, Davis and I have predicted, and then confirmedexperimentally, (i) the area-invariance of deep-germ-layer surfacetensions in vitro, (ii) spontaneous cell slippage in deformeddeep—germ—layer cell aggregates and (iii) correlationsof tissue surface tensions with tissue positioning in deep-germ-layercell-sorting and aggregate—fusion experiments. Liquid—tissueflow involves intercalations of interior cells into expandingtissue interfaces (or withdrawal of surface cells from shrinkingtissue interfaces). Tissue surface tensions are macroscopicreflections of the microscopic, tissue—specific adhesivedifferentials which direct these cell translocations. Such adhesivedifferentials may act independently of, or together with, activecell—shape changes, chemotaxis, contact guidance and/orhaptotaxis in controlling embryonic tissue rearrangements. Deepcell intercalations in vivo occur throughout amphibian gastrulation:during ectodermal epiboly; during marginal—zone extensionand convergence (and therefore blastopore closure); during mesodermalinvolution; and probably during the anterior spreading and axialextension (and therefore dorsal convergence) of involuted mesoderm.Tissue—surface—tension measurements may help determine(i) which of these cell—intercalation processes are activeand which are passive, (ii) the specific contributions of variousmicroscopic cell properties to the regulation of liquid—likegerm—layer assembly and (iii) similarities and differencesbetween in vitro and in vivo control mechanisms governing amphibiangastrulation.     

WallGen,Software to Construct Layered Cellulose-Hemicellulose Networks and Predict Their Small Deformation Mechanics

We understand few details about how the arrangement and interactions of cell wall polymers produce the mechanical properties of primary cell walls. Consequently, we cannot quantitatively assess if proposed wall structures are mechanically reasonable or assess the effectiveness of proposed mechanisms to change mechanical properties. As a step to remedying this, we developed WallGen, a Fortran program (available on request) building virtual cellulose-hemicellulose networks by stochastic self-assembly whose mechanical properties can be predicted by finite element analysis. The thousands of mechanical elements in the virtual wall are intended to have one-to-one spatial and mechanical correspondence with their real wall counterparts of cellulose microfibrils and hemicellulose chains. User-defined inputs set the properties of the two polymer types (elastic moduli, dimensions of microfibrils and hemicellulose chains, hemicellulose molecular weight) and their population properties (microfibril alignment and volume fraction, polymer weight percentages in the network). This allows exploration of the mechanical consequences of variations in nanostructure that might occur in vivo and provides estimates of how uncertainties regarding certain inputs will affect WallGen''s mechanical predictions. We summarize WallGen''s operation and the choice of values for user-defined inputs and show that predicted values for the elastic moduli of multinet walls subject to small displacements overlap measured values. “Design of experiment” methods provide systematic exploration of how changed input values affect mechanical properties and suggest that changing microfibril orientation and/or the number of hemicellulose cross-bridges could change wall mechanical anisotropy.Plant scientists have long studied how primary wall structure influences mechanical properties (Preston, 1974). In this work, we develop methods to predict the elastic modulus for layered networks of cellulose microfibrils (CMFs) cross-linked by hemicellulose (HC) chains when they are subject to small imposed displacements.Polysaccharides provide over 90% of wall mass and therefore are likely to dominate wall mechanics. Two distinct but probably interacting (Zykwinska et al., 2005) networks are recognized: a cellulose-hemicellulose (CHC) network and a pectin network. Pectins can be removed by mutations, allowing measurements of the mechanical properties of the CHC network (Ryden et al., 2003) that can be compared with predicted values. The two networks probably make roughly comparable mechanical contributions in pectin-rich dicots (Ryden et al., 2003), but the CHC network presumably dominates in monocots with pectin-poor, type II walls (Carpita and Gibeaut, 1993; Rose, 2003). Plant cells align CMFs (Baskin, 2005) but not noncellulosic polysaccharides such as pectins and HCs. CMF alignment, therefore, underlies the structural and mechanical anisotropy seen in many cell walls.In principle, wall structure can predict mechanical properties, a multiscale modeling problem of the type that materials scientists often tackle (Kwon et al., 2008). In this context, structural and mechanical inputs concern polymer chains or aggregates, and mechanical properties are predicted for pieces of material several orders of magnitude larger that contain many polymer chains. There are some structure-based quantitative predictions of the mechanics of secondary walls (Bergander and Salmén, 2002; Keckes et al., 2003; Salmén, 2004; Hofstetter et al., 2005; Altaner and Jarvis, 2008), but most discussions of primary walls only involve qualitative consideration of how factors such as CMF length and alignment might change growth anisotropy (Wasteneys, 2004; Baskin, 2005) rather than the small displacement mechanical properties with which we are concerned. Modeling plant cell walls provides several particular challenges. First, walls vary greatly in CMF alignment, with multinet, polylamellate, helicoidal, and other types recognized; second, polymer composition varies even within one wall type; and third, polymer interactions remain uncertain, with the view that HCs cross-bridge CMFs (Hayashi, 1989) challenged on various grounds by those regarding them as providing spacing or otherwise facilitating movement between CMFs (Whitney et al., 1999; Thompson, 2005). In beginning multiscale modeling of primary walls, therefore, we sought a strategy that facilitated in silico experiments in which we could vary the structure, composition, and other wall properties that contribute to the complex microstructure of cell walls and that provided the opportunity to give the polymers more complex properties in future studies.Many modeling strategies facilitate computation by simplifying (homogenizing) wall structure by aggregating the properties of many polymers. Procedures are well established but do not obviate the necessity to understand the underlying polymer properties and impose an additional requirement to deduce the properties of the population being homogenized. Cell walls are often compared with fiber composites, for which several approaches to the prediction of the elastic properties have been reported (Chamis and Sendeckyj, 1968). Most such micromechanical approaches, however, are based on simplifying assumptions about the geometry of the microstructure or special relations between the phase properties. Moreover, although cell walls are often described as fiber composites, this obscures important distinctions, notably the difference between the continuous interfiber matrix typical of most manufactured fiber composites and the discrete HC cross-links present in the cell wall. The mechanical properties of the continuous matrix are relatively easily measured for manufactured fiber composites, but replacing HC cross-bridges with a continuous matrix requires defining its mechanical properties. Various micromechanical models of secondary walls assume that a homogenous HC matrix surrounds CMFs (Bergander and Salmén, 2002; Salmén, 2004; Hofstetter et al., 2005); for example, Hofstetter et al. (2005) gave this phase a bulk modulus taken from testing an isotropic HC powder. Increased computing power now provides the option to avoid such homogenization with at least three advantages accruing. First, homogenization often limits the ease with which different structures can be investigated (a high priority issue for us), since homogenization assumptions may need to be reexamined and recalibrated as microstructure changes. Without homogenization, a wide range of structures can be analyzed, given that a flexible system is available for generating microstructure. Second, accumulating knowledge of the mechanics of individual polymer chains coming from techniques such as atomic force microscopy can be directly applied to the individual HCs and CMFs in a nonhomogenized model. If homogenization is applied, that relationship is lost and new assumptions must be made about the properties of the population. Third, once the basic model is established, the properties of the polymers, particularly those of the HCs, can be varied to more accurately capture the nonlinear and other properties seen on extension.We avoided homogenization by using the WallGen program to build a fragment of virtual wall whose components have one-to-one spatial and mechanical correspondence with the CMFs and HCs of a primary wall CHC network. We chose finite element analysis (FEA) to predict the mechanical properties of the entire fragment containing thousands of CMFs and HCs. In effect, then, WallGen averages by setting up the most realistic spatial arrangement, using mechanical data for individual chains and leaving FEA to predict the collective properties. The well-established engineering technique of FEA has been used to predict wall mechanics at cellular and subcellular scales. Examples include predicting cell response to microindentation (Bolduc et al., 2006) or compression between flat plates (Smith et al., 1998) and predicting the mechanics of pulped fiber networks in paper (Hansson and Rasmuson, 2004). These applications have not involved mechanical representation of individual wall polymers, but FEA has been used at this scale to model individual microtubules and F-actin polymers pulling on membranes (Allen et al., 2009) and at even finer scales to model tubulin lattice deformation within single microtubules (Schaap et al., 2006). Modern FEA programs have features of potential value for developing more sophisticated models of wall mechanics: components can have nonlinear force-extension properties and viscoelastic properties, and conditions can be specified to break links between components of the microstructure. This should allow exploration of the more complex mechanical behavior that CHC networks show when subject to larger displacements and incorporation of additional mechanical elements providing the properties generated by pectins.In this article, we describe how WallGen operates, review the choice of values for several important inputs, predict the elastic moduli of multinet walls in which HCs cross-link CMFs, compare those values with experimental values, and quantify the mechanical effects of varying several inputs to the virtual wall. We restrict consideration to polymers given linear elastic properties and, because small strains are sufficient to predict the elastic modulus, restrict experiments to small displacements to minimize inaccuracies from this simplification. A previous publication considered issues relating to representative volume elements and analyzed some simpler CHC networks (Kha et al., 2008).     

Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.     

基于测序软件进行生物信息学中数据分析

概述了生物信息学中的一些研究方向及分析方法,介绍了用于大规模DNA测序的分析软件系统——Phred/Phrap/Consed。通过利用Phred/Phrap/Consed等各种分析软件,时基因组学、蛋白质组学和基因芯片研究中巨量原始实验数据进行分析、处理,使之成为具有明确生物学意义的生物信息。     

Transport of Compatible Solutes in Extremophiles

Salt-tolerant as well as moderately halophilic and halophilic organisms have to maintain their turgor. One strategy is to accumulate small organic compounds, compatible solutes, by de novo synthesis or uptake. From a bioenergetic point of view, uptake is preferred over biosynthesis. The transport systems catalyzing uptake of compatible solutes are of primary or secondary nature and coupled to ATP hydrolysis or ion (H+, Na+) symport. Expression of the transporter genes as well as the activity of the transporters is regulated by salinity/osmolarity and one of the key questions is how salinity or osmolarity is sensed and the signal transmitted as far as to gene expression and transporter activation. Recent studies shed light on the nature and the activation mechanisms of solute transporters in extremophiles, and this review summarizes current knowledge on the structure, function and osmo- or salt-regulation of transporters for compatible solutes in extremophiles.     

TRANSIT - A Software Tool for Himar1 TnSeq Analysis

TnSeq has become a popular technique for determining the essentiality of genomic regions in bacterial organisms. Several methods have been developed to analyze the wealth of data that has been obtained through TnSeq experiments. We developed a tool for analyzing Himar1 TnSeq data called TRANSIT. TRANSIT provides a graphical interface to three different statistical methods for analyzing TnSeq data. These methods cover a variety of approaches capable of identifying essential genes in individual datasets as well as comparative analysis between conditions. We demonstrate the utility of this software by analyzing TnSeq datasets of M. tuberculosis grown on glycerol and cholesterol. We show that TRANSIT can be used to discover genes which have been previously implicated for growth on these carbon sources. TRANSIT is written in Python, and thus can be run on Windows, OSX and Linux platforms. The source code is distributed under the GNU GPL v3 license and can be obtained from the following GitHub repository: https://github.com/mad-lab/transit

This is a PLOS Computational Biology Software paper

     

Compatible host/mycorrhizal fungus combinations for micropropagated sea oats

Micropropagation technology promises to improve the supply of sea oats for restoring Florida's eroded beaches, but concerns about genetic diversity need to be addressed. These dune plants are colonized by a wide array of arbuscular mycorrhizal (AM) fungi, yet little is know of the diversity of these fungal communities. Our goal was to test the level of functional diversity that exists among communities of AM fungi that are present in divergent Florida dunes. Community pot cultures were established from samples collected from ten transects in two Gulf coast and two Atlantic coast locations in Florida, and these were used to conduct two greenhouse studies. The objective of the first study was to evaluate within-location variance in the mycorrhizal function of different AM fungal communities associated with endemic sea oats. The objective of the second study was to evaluate among-location responses of plant and fungal ecotypes using selected combinations obtained from the first experiment. Within locations, the AM fungal community had significant impacts on shoot mass and shoot-P contents, confirming a range of symbiotic effectiveness exists within the beach-dune system. Among locations, there was a tendency for greater root colonization between host clones and fungal communities from the same location, indicating a degree of specificity between host ecotypes and their symbiotic fungi. Relative to plant growth response, one fungal community was superior across plant genotypes from all locations, while one plant genotype tended to have the best response across all fungal communities. These data suggest that while it is possible to select effective AM fungal-host combinations for outplanting, origin of host and AM fungi have little predictive value in screening these combinations.     

BEAST 2: A Software Platform for Bayesian Evolutionary Analysis

We present a new open source, extensible and flexible software platform for Bayesian evolutionary analysis called BEAST 2. This software platform is a re-design of the popular BEAST 1 platform to correct structural deficiencies that became evident as the BEAST 1 software evolved. Key among those deficiencies was the lack of post-deployment extensibility. BEAST 2 now has a fully developed package management system that allows third party developers to write additional functionality that can be directly installed to the BEAST 2 analysis platform via a package manager without requiring a new software release of the platform. This package architecture is showcased with a number of recently published new models encompassing birth-death-sampling tree priors, phylodynamics and model averaging for substitution models and site partitioning. A second major improvement is the ability to read/write the entire state of the MCMC chain to/from disk allowing it to be easily shared between multiple instances of the BEAST software. This facilitates checkpointing and better support for multi-processor and high-end computing extensions. Finally, the functionality in new packages can be easily added to the user interface (BEAUti 2) by a simple XML template-based mechanism because BEAST 2 has been re-designed to provide greater integration between the analysis engine and the user interface so that, for example BEAST and BEAUti use exactly the same XML file format.

This is a PLOS Computational Biology Software Article.

     

Determining Protein-Induced DNA Bending in Force-Extension Experiments: Theoretical Analysis

Computer simulations were used to investigate the possibility of determining protein-induced DNA bend angles by measuring the extension of a single DNA molecule. Analysis of the equilibrium sets of DNA conformations showed that shortening of DNA extension by a single protein-induced DNA bend can be as large as 35 nm. The shortening has a maximum value at the extending force of ∼0.1 pN. At this force, the DNA extension experiences very large fluctuations that dramatically complicate the measurement. Using Brownian dynamics simulation of a DNA molecule extended by force, we were able to estimate the observation time needed to obtain the desired accuracy of the extension measurement. Also, the simulation revealed large fluctuations of the force, acting on the attached magnetic bead from the stretched DNA molecule.     

Electronic Structure Analysis of a New Quinoid Conjugated Polymer

The low lying unoccupied orbitals of oligomers of 4-dicyanomethylene-4H-cyclopenta[2,1-b:3,4-b'] dithiophene (CDM) are not delocalized over the whole molecule. Is such electron localization in the conduction band of poly-CDM responsible for its low n-type conductivity? Are polymers of the tricyclic thioketone (TCT) with more delocalized unoccupied orbitals a better alternative for stable n-dopable conducting polymers? Monomer through tetramer of TCT have been optimized with density functional theory. IP, EA, energy gap, and band width of the corresponding polymer were obtained by extrapolation. Comparison with data for oligomers of 4-dicyanomethylene-4H-cyclopenta[2,1-b:3,4-b'] dithiophene and of thiophene indicates that the novel polymer would have a small band gap and would fulfil the conditions for n-dopability and high mobility of n-type carriers.     

Analysis of Quantum Mechanics Microscopic Mechanism on Genetic Mutagenesis of Laser

用量子力学研究了激光对ＤＮＡ的键（或链）的局域相互作用及激光对ＤＮＡ分子系统的相互作用的微观机理。研究结果可以解释激光的遗传诱变效应。     

Polymer support for exonucleolytic sequencing

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene "core" and a "shell" of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 microm diameter. Applying M13 ssDNA in extremely high dilution (approximately 1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 microm diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand.     

Mechanics of microtubules

Microtubules are rigid cytoskeletal filaments, and their mechanics affect cell morphology and cellular processes. For instance, microtubules for the support structures for extended morphologies, such as axons and cilia. Further, microtubules act as tension rods to pull apart chromosomes during cellular division. Unlike other cytoskeletal filaments (e.g., actin) that work as large networks, microtubules work individually or in small groups, so their individual mechanical properties are quite important to their cellular function. In this review, we explore the past work on the mechanics of individual microtubules, which have been studied for over a quarter of a century. We also present some prospective on future endeavors to determine the molecular mechanisms that control microtubule rigidity.