WorldWideScience

Sample records for delta-endotoxin complex revealed

  1. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into... Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens is exempt from...

  2. Experimental design and Bayesian networks for enhancement of delta-endotoxin production by Bacillus thuringiensis.

    Science.gov (United States)

    Ennouri, Karim; Ayed, Rayda Ben; Hassen, Hanen Ben; Mazzarello, Maura; Ottaviani, Ennio

    2015-12-01

    Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH₂PO₄, K₂HPO₄, FeSO₄, MnSO₄, and MgSO₄and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett-Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO₄, K2HPO₄, starch and soybean meal. Indeed, it was found that soybean meal, K₂HPO₄, KH₂PO₄and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett-Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation.

  3. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated... of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens...

  4. Introduction of Delta-endotoxin Gene of Bacillus thuringiensis to Chrysanthemum [Dendranthema grandiflorum(Ramat.) Kitamura] for Insect Resistance

    National Research Council Canada - National Science Library

    Shinoyama, Harue; Komano, Masayasu; Nomura, Yukio; Nagai, Teruyuki

    2002-01-01

    We introduced a truncated delta-endotoxin gene, cry1Ab of Bacillus thuringiensis which has a specific biological activity against lepidopteran insects into chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura...

  5. Mathematical relationships between spore concentrations, delta-endotoxin levels, and entomotoxicity of Bacillus thuringiensis preparations produced in different fermentation media.

    Science.gov (United States)

    Vu, Khanh Dang; Tyagi, R D; Surampalli, R Y; Valéro, J R

    2012-11-01

    Mathematic relationships between spore concentrations, delta-endotoxin concentrations and entomotoxicity (Tx) of Bacillus thuringiensis var. kurstaki HD-1 (Btk HD-1) preparations produced in six different media were analysed. The relationship between delta-endotoxin and spore concentration and SpTx-spore (specific Tx per 1000 spore) and spore concentration produced in the different media (starch industry wastewater (SIW) with total solids (TS) concentration of 15g/L, SIW with TS of 30g/L, SIW supplemented with 0.2% (w/v) colloidal chitin, SIW supplemented with 1.25% (w/v) cornstarch and 0.2% (v/v) Tween 80, secondary sludge, and semi-synthetic medium) strictly followed the Power law. Tx and delta endotoxin concentration followed the exponential relation whereas a definite relation between Tx and spore concentration could not be established. Spore and delta-endotoxin produced at the early time (12h) during fermentation might be more toxic than those produced during latter period of fermentation irrespective of media used. Tx and delta-endotoxin concentration exhibited a semi-log linear relationship. Based on these findings, delta-endotoxin concentration can be determined rapidly to monitor the progress of the biopesticide production process.

  6. Structural studies of {delta}-endotoxin Cry 1 C from Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Guimaraes, B.G.; Garratt, R.C.; Oliva, G. [Sao Paulo Univ., Sao Carlos, SP (Brazil). Inst. de Fisica; Lemos, M.V.F. [UNESP, Jaboticabal, SP (Brazil). Dept. de Biologia Aplicada Agropecuaria; Arantes, O.M.N. [Universidade Estadual de Londrina, PR (Brazil). Dept. de Biologia Geral

    1996-12-31

    Full text. The {delta}-endotoxins are a family of crystal protein by a soil bacterium, Bacillus thuringiensis. The study of these proteins has been of great interest due to their highly specific activity against insects of the orders Lepidoptera, Diptera and Coleoptera. Thus, the {delta}a-endotoxins have been used for more than two decades as biological insecticides to control agricultural pests and, more recently, insects vectors of some diseases. The knowledge of their three-dimensional structures is very important to understand their mechanism of action and their high specificity. To date, the structure of only three proteins of the {delta}-endotoxins family have been reported: Cry3A, a coleopteran-specific toxin (beetle toxin){sup 1}, Cry1Aa, a lepidopteran-specific toxin (butterfly toxin){sup 2} and CytB, a dipteran-specific toxin (mosquito toxin){sup 3} Our work is aimed at the determination of the crystallographic structure by X-ray diffraction of {delta}-endotoxin Cry1C, also toxic to insects of the Lepidoptera order but towards families other than those affected by Cry1Aa. A comparison between these structures may lead to important conclusions about the reasons for the specificity and would allow the planning of mutants with more efficient activity. The cry1C gene was cloned into an adequate vector and expressed in an acrystalliferous B. thuringiensis strain. After cell culture and sporulation the microcrystals of Cry1C were separated by ultra-centrifugation in sacharose. The protoxin inclusion bodies were activated by commercial trpsin and the protease-resistant core was purified by anion-exchange chromatography. Crystallization experiments are being conducted in order to obtain single crystals suitable for diffraction measurements. We intend to use the Protein Crystallograph Station of the LNLS to collect data as soon as it is available and we have suitable crystals. (author) 3 refs.

  7. A hybrid Bacillus thuringiensis delta-endotoxin gene gives resistance against a coleopteran and a lepidopteran pest in transgenic potato

    NARCIS (Netherlands)

    Naimov, S.; Dukiandjiev, S.; Maagd, de R.A.

    2003-01-01

    Expression of Bacillus thuringiensis delta-endotoxins has proven to be a successful strategy for obtaining insect resistance in transgenic plants. Drawbacks of expression of a single resistance gene are the limited target spectrum and the potential for rapid adaptation of the pest. Hybrid toxins wit

  8. Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle

    NARCIS (Netherlands)

    Naimov, S.; Weemen-Hendriks, M.; Dukiandjiev, S.; Maagd, de R.A.

    2001-01-01

    Cry1 delta-endotoxins of Bacillus thuringiensis are generally active against lepidopteran insects, but Cry1Ba and Cry1Ia have additional, though low, levels of activity against coleopterans such as the Colorado potato beetle. Here we report the construction of Cry1Ba/Cry1Ia hybrid toxins which have

  9. Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis.

    Science.gov (United States)

    Ghribi, D; Zouari, N; Jaoua, S

    2004-01-01

    The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in delta-endotoxin synthesis. Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of delta-endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of delta-endotoxin synthesis. Classical mutagenesis of Bt cells lead to the isolation of a large variety of delta-endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and delta-endotoxin synthesis. High frequencies of delta-endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.

  10. Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae

    Directory of Open Access Journals (Sweden)

    CFG Cavados

    2004-08-01

    Full Text Available The bacterium Bacillus thuringiensis (Bt produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584, serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

  11. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Science.gov (United States)

    2010-07-01

    ...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the expression plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  12. Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

    Science.gov (United States)

    Nayak, P; Basu, D; Das, S; Basu, A; Ghosh, D; Ramakrishnan, N A; Ghosh, M; Sen, S K

    1997-03-18

    Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

  13. Revealing the Hidden Language of Complex Networks

    Science.gov (United States)

    Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Davis, Darren; Levnajic, Zoran; Janjic, Vuk; Karapandza, Rasa; Stojmirovic, Aleksandar; Pržulj, Nataša

    2014-04-01

    Sophisticated methods for analysing complex networks promise to be of great benefit to almost all scientific disciplines, yet they elude us. In this work, we make fundamental methodological advances to rectify this. We discover that the interaction between a small number of roles, played by nodes in a network, can characterize a network's structure and also provide a clear real-world interpretation. Given this insight, we develop a framework for analysing and comparing networks, which outperforms all existing ones. We demonstrate its strength by uncovering novel relationships between seemingly unrelated networks, such as Facebook, metabolic, and protein structure networks. We also use it to track the dynamics of the world trade network, showing that a country's role of a broker between non-trading countries indicates economic prosperity, whereas peripheral roles are associated with poverty. This result, though intuitive, has escaped all existing frameworks. Finally, our approach translates network topology into everyday language, bringing network analysis closer to domain scientists.

  14. Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

    Directory of Open Access Journals (Sweden)

    Aamena Alshamsi

    Full Text Available Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

  15. Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

    Science.gov (United States)

    Gong, Chaohui; Astley, Henry; Schiebel, Perrin; Dai, Jin; Travers, Matthew; Goldman, Daniel; Choset, Howie; CMU Team; GT Team

    Geometric mechanics offers useful tools for intuitively analyzing biological and robotic locomotion. However, utility of these tools were previously restricted to systems that have only two internal degrees of freedom and in uniform media. We show kinematics of complex locomotors that make intermittent contacts with substrates can be approximated as a linear combination of two shape bases, and can be represented using two variables. Therefore, the tools of geometric mechanics can be used to analyze motions of locomotors with many degrees of freedom. To demonstrate the proposed technique, we present studies on two different types of snake gaits which utilize combinations of waves in the horizontal and vertical planes: sidewinding (in the sidewinder rattlesnake C. cerastes) and lateral undulation (in the desert specialist snake C. occipitalis). C. cerastes moves by generating posteriorly traveling body waves in the horizontal and vertical directions, with a relative phase offset equal to +/-π/2 while C. occipitalismaintains a π/2 offset of a frequency doubled vertical wave. Geometric analysis reveals these coordination patterns enable optimal movement in the two different styles of undulatory terrestrial locomotion. More broadly, these examples demonstrate the utility of geometric mechanics in analyzing realistic biological and robotic locomotion.

  16. Intersubject information mapping: revealing canonical representations of complex natural stimuli

    Directory of Open Access Journals (Sweden)

    Nikolaus Kriegeskorte

    2015-03-01

    Full Text Available Real-world time-continuous stimuli such as video promise greater naturalism for studies of brain function. However, modeling the stimulus variation is challenging and introduces a bias in favor of particular descriptive dimensions. Alternatively, we can look for brain regions whose signal is correlated between subjects, essentially using one subject to model another. Intersubject correlation mapping (ICM allows us to find brain regions driven in a canonical manner across subjects by a complex natural stimulus. However, it requires a direct voxel-to-voxel match between the spatiotemporal activity patterns and is thus only sensitive to common activations sufficiently extended to match up in Talairach space (or in an alternative, e.g. cortical-surface-based, common brain space. Here we introduce the more general approach of intersubject information mapping (IIM. For each brain region, IIM determines how much information is shared between the subjects' local spatiotemporal activity patterns. We estimate the intersubject mutual information using canonical correlation analysis applied to voxels within a spherical searchlight centered on each voxel in turn. The intersubject information estimate is invariant to linear transforms including spatial rearrangement of the voxels within the searchlight. This invariance to local encoding will be crucial in exploring fine-grained brain representations, which cannot be matched up in a common space and, more fundamentally, might be unique to each individual – like fingerprints. IIM yields a continuous brain map, which reflects intersubject information in fine-grained patterns. Performed on data from functional magnetic resonance imaging (fMRI of subjects viewing the same television show, IIM and ICM both highlighted sensory representations, including primary visual and auditory cortices. However, IIM revealed additional regions in higher association cortices, namely temporal pole and orbitofrontal cortex. These

  17. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. Copyright © 2015, American Association for the Advancement of Science.

  18. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    Directory of Open Access Journals (Sweden)

    Gustafsson Lars

    2010-12-01

    Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

  19. New burgess shale fossil sites reveal middle cambrian faunal complex.

    Science.gov (United States)

    Collins, D; Briggs, D; Morris, S C

    1983-10-14

    Soft-bodied and lightly sclerotized Burgess shale fossils have been found at more than a dozen new localities in an area extending for 20 kilometers along the front of the Cathedral Escarpment in the Middle Cambrian Stephen Formation of the Canadian Rockies. Five different fossil assemblages from four stratigraphic levels have been recognized. These assemblages represent distinct penecontemporaneous marine communities that together make up a normal fore-reef faunal complex.

  20. Maps of random walks on complex networks reveal community structure.

    Science.gov (United States)

    Rosvall, Martin; Bergstrom, Carl T

    2008-01-29

    To comprehend the multipartite organization of large-scale biological and social systems, we introduce an information theoretic approach that reveals community structure in weighted and directed networks. We use the probability flow of random walks on a network as a proxy for information flows in the real system and decompose the network into modules by compressing a description of the probability flow. The result is a map that both simplifies and highlights the regularities in the structure and their relationships. We illustrate the method by making a map of scientific communication as captured in the citation patterns of >6,000 journals. We discover a multicentric organization with fields that vary dramatically in size and degree of integration into the network of science. Along the backbone of the network-including physics, chemistry, molecular biology, and medicine-information flows bidirectionally, but the map reveals a directional pattern of citation from the applied fields to the basic sciences.

  1. Hierarchicality of trade flow networks reveals complexity of products.

    Science.gov (United States)

    Shi, Peiteng; Zhang, Jiang; Yang, Bo; Luo, Jingfei

    2014-01-01

    With globalization, countries are more connected than before by trading flows, which amounts to at least 36 trillion dollars today. Interestingly, around 30-60 percents of exports consist of intermediate products in global. Therefore, the trade flow network of particular product with high added values can be regarded as value chains. The problem is weather we can discriminate between these products from their unique flow network structure? This paper applies the flow analysis method developed in ecology to 638 trading flow networks of different products. We claim that the allometric scaling exponent η can be used to characterize the degree of hierarchicality of a flow network, i.e., whether the trading products flow on long hierarchical chains. Then, it is pointed out that the flow networks of products with higher added values and complexity like machinary, transport equipment etc. have larger exponents, meaning that their trade flow networks are more hierarchical. As a result, without the extra data like global input-output table, we can identify the product categories with higher complexity, and the relative importance of a country in the global value chain by the trading network solely.

  2. Hierarchicality of trade flow networks reveals complexity of products.

    Directory of Open Access Journals (Sweden)

    Peiteng Shi

    Full Text Available With globalization, countries are more connected than before by trading flows, which amounts to at least 36 trillion dollars today. Interestingly, around 30-60 percents of exports consist of intermediate products in global. Therefore, the trade flow network of particular product with high added values can be regarded as value chains. The problem is weather we can discriminate between these products from their unique flow network structure? This paper applies the flow analysis method developed in ecology to 638 trading flow networks of different products. We claim that the allometric scaling exponent η can be used to characterize the degree of hierarchicality of a flow network, i.e., whether the trading products flow on long hierarchical chains. Then, it is pointed out that the flow networks of products with higher added values and complexity like machinary, transport equipment etc. have larger exponents, meaning that their trade flow networks are more hierarchical. As a result, without the extra data like global input-output table, we can identify the product categories with higher complexity, and the relative importance of a country in the global value chain by the trading network solely.

  3. Revealing the macromolecular targets of complex natural products

    Science.gov (United States)

    Reker, Daniel; Perna, Anna M.; Rodrigues, Tiago; Schneider, Petra; Reutlinger, Michael; Mönch, Bettina; Koeberle, Andreas; Lamers, Christina; Gabler, Matthias; Steinmetz, Heinrich; Müller, Rolf; Schubert-Zsilavecz, Manfred; Werz, Oliver; Schneider, Gisbert

    2014-12-01

    Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product ‘de-orphaning’.

  4. The Capsaspora genome reveals a complex unicellular prehistory of animals.

    Science.gov (United States)

    Suga, Hiroshi; Chen, Zehua; de Mendoza, Alex; Sebé-Pedrós, Arnau; Brown, Matthew W; Kramer, Eric; Carr, Martin; Kerner, Pierre; Vervoort, Michel; Sánchez-Pons, Núria; Torruella, Guifré; Derelle, Romain; Manning, Gerard; Lang, B Franz; Russ, Carsten; Haas, Brian J; Roger, Andrew J; Nusbaum, Chad; Ruiz-Trillo, Iñaki

    2013-01-01

    To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans' unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans.

  5. Revealing the complex conduction heat transfer mechanism of nanofluids.

    Science.gov (United States)

    Sergis, A; Hardalupas, Y

    2015-12-01

    Nanofluids are two-phase mixtures consisting of small percentages of nanoparticles (sub 1-10 %vol) inside a carrier fluid. The typical size of nanoparticles is less than 100 nm. These fluids have been exhibiting experimentally a significant increase of thermal performance compared to the corresponding carrier fluids, which cannot be explained using the classical thermodynamic theory. This study deciphers the thermal heat transfer mechanism for the conductive heat transfer mode via a molecular dynamics simulation code. The current findings are the first of their kind and conflict with the proposed theories for heat transfer propagation through micron-sized slurries and pure matter. The authors provide evidence of a complex new type of heat transfer mechanism, which explains the observed abnormal heat transfer augmentation. The new mechanism appears to unite a number of popular speculations for the thermal heat transfer mechanism employed by nanofluids as predicted by the majority of the researchers of the field into a single one. The constituents of the increased diffusivity of the nanoparticle can be attributed to mismatching of the local temperature profiles between parts of the surface of the solid and the fluid resulting in increased local thermophoretic effects. These effects affect the region surrounding the solid manifesting interfacial layer phenomena (Kapitza resistance). In this region, the activity of the fluid and the interactions between the fluid and the nanoparticle are elevated. Isotropic increased nanoparticle mobility is manifested as enhanced Brownian motion and diffusion effects.

  6. Communities in Neuronal Complex Networks Revealed by Activation Patterns

    CERN Document Server

    Costa, Luciano da Fontoura

    2008-01-01

    Recently, it has been shown that the communities in neuronal networks of the integrate-and-fire type can be identified by considering patterns containing the beginning times for each cell to receive the first non-zero activation. The received activity was integrated in order to facilitate the spiking of each neuron and to constrain the activation inside the communities, but no time decay of such activation was considered. The present article shows that, by taking into account exponential decays of the stored activation, it is possible to identify the communities also in terms of the patterns of activation along the initial steps of the transient dynamics. The potential of this method is illustrated with respect to complex neuronal networks involving four communities, each of a different type (Erd\\H{o}s-R\\'eny, Barab\\'asi-Albert, Watts-Strogatz as well as a simple geographical model). Though the consideration of activation decay has been found to enhance the communities separation, too intense decays tend to y...

  7. TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

    Institute of Scientific and Technical Information of China (English)

    Hua-rong,Li; BrendaOppert; KunYanZhu; RandallA.Higgins; Fang-nengHuang; LawrentL.Buschman

    2003-01-01

    Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modem high-expression transgenic Bt plants possible, with an emphasis on the reasons for the low-level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.

  8. Conjunctival lymphangioma in a 4-year-old girl revealed tuberous sclerosis complex

    Directory of Open Access Journals (Sweden)

    Freiberg, Florentina Joyce

    2016-09-01

    Full Text Available Background: To present a case of conjunctival lymphangioma in a girl with tuberous sclerosis complex.Methods/results: A 4-year-old girl presented with a relapsing cystic lesion of the bulbar conjunctiva in the right eye with string-of-pearl-like dilation of lymphatic vessels and right-sided facial swelling with mild pain. Best-corrected vision was not impaired. Examination of the skin revealed three hypomelanotic macules and a lumbal Shagreen patch. Magnetic resonance imaging (MRI findings displayed minimal enhancement of buccal fat on the right side. Cranial and orbital MRI showed signal enhancement in the right cortical and subcortical areas. Genetic analysis revealed a heterozygous deletion encompassing exon 1 and 2 of the gene (tuberous sclerosis complex 1 gene, confirming the diagnosis of tuberous sclerosis complex.Conclusion: In conjunctival lymphangioma, tuberous sclerosis complex should be considered as the primary disease.

  9. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

    DEFF Research Database (Denmark)

    Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

    2011-01-01

    with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only...

  10. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.

    Directory of Open Access Journals (Sweden)

    Mariya M Kucherenko

    Full Text Available The Dystroglycan-Dystrophin (Dg-Dys complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.

  11. Concordance of gene expression in human protein complexes reveals tissue specificity and pathology

    DEFF Research Database (Denmark)

    Börnigen, Daniela; Pers, Tune Hannes; Thorrez, Lieven

    2013-01-01

    Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among...... proteins in a complex within a given tissue may pinpoint tissues that will be affected by a mutation in the complex and coordinated expression may reveal the complex to be active in the tissue. We identified known disease genes and their protein complex partners in a high-quality human interactome. Each...... susceptibility gene's tissue involvement was ranked based on coordinated expression with its interaction partners in a non-disease global map of human tissue-specific expression. The approach demonstrated high overall area under the curve (0.78) and was very successfully benchmarked against a random model...

  12. Genome evolution predicts genetic interactions in protein complexes and reveals cancer drug targets

    NARCIS (Netherlands)

    Lu, X.; Kensche, P.R.; Huynen, M.A.; Notebaart, R.A.

    2013-01-01

    Genetic interactions reveal insights into cellular function and can be used to identify drug targets. Here we construct a new model to predict negative genetic interactions in protein complexes by exploiting the evolutionary history of genes in parallel converging pathways in metabolism. We evaluate

  13. Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex.

    Science.gov (United States)

    Vukotic, Milena; Oeljeklaus, Silke; Wiese, Sebastian; Vögtle, F Nora; Meisinger, Chris; Meyer, Helmut E; Zieseniss, Anke; Katschinski, Doerthe M; Jans, Daniel C; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter; Deckers, Markus

    2012-03-01

    The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

  14. Distinct levels in the nanoscale organization of DNA-histone complex revealed by its mechanical unfolding

    Science.gov (United States)

    Soni, G. V.; Brar, Loveleen; Hameed, Feroz M.; Raychaudhuri, A. K.; Shivashankar, G. V.

    2007-04-01

    Mechanical unfolding of nanoscale DNA-histone complex, using an atomic force microscope, shows a stepwise disassembly of histones from the nucleosome. A quantitative analysis of the rupture jump statistics and the length released per jump reveals insights into the possible histone contacts within the octamer complex. The measured ruptures correlate with the breakage of multiple contacts that stabilize the histone octamer. These results provide a mechanistic basis by which stepwise disassembly of histone proteins may result from an external force exerted by the adenosinetriphosphate (ATP) dependent chromatin remodeling machines to access regulatory sites on DNA.

  15. Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist.

    Science.gov (United States)

    Törnroth-Horsefield, Susanna; Gourdon, Pontus; Horsefield, Rob; Brive, Lars; Yamamoto, Natsuko; Mori, Hirotada; Snijder, Arjan; Neutze, Richard

    2007-12-01

    Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

  16. Information processing reveals how microscopic components affect the macroscopic system-state in complex networks

    CERN Document Server

    Quax, Rick; Sloot, Peter M A

    2013-01-01

    Nature processes information. We observe this from physical systems, which register information in the system state, transfer information through interactions, and lose information due to thermal noise. Being able to quantify this information processing could lead to a unifying framework for a better understanding of complex systems. In this letter we present a formalism to describe to what extent a macroscopic system is affected by the microstates of its constituents. We study this numerically for a scale-free network of Ising-spins, a prototypical complex system, and present an answer to the unexplained phenomenon that real systems with a heterogeneous topology are mainly controlled by nodes with fewer connections. Counter to intuition we find that due to selective information dissipation, not the hubs but rather the intermediately connected nodes are remembered best by the system. Our study reveals that the framework of information processing improves our understanding of complex systems at large.

  17. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    Science.gov (United States)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  18. Kinetic analyses reveal multiple steps in forming TonB-FhuA complexes from Escherichia coli.

    Science.gov (United States)

    Khursigara, Cezar M; De Crescenzo, Gregory; Pawelek, Peter D; Coulton, James W

    2005-03-08

    FhuA, an outer membrane receptor of Escherichia coli, facilitates transport of hydroxamate siderophores and siderophore-antibiotic conjugates. The cytoplasmic membrane complex TonB-ExbB-ExbD provides energy for transport via the proton motive force. This energy is transduced by protein-protein interactions between TonB and FhuA, but the molecular determinants of these interactions remain uncharacterized. Our analyses of FhuA and two recombinant TonB species by surface plasmon resonance revealed that TonB undergoes a kinetically limiting rearrangement upon initial interaction with FhuA: an intermediate TonB-FhuA complex of 1:1 stoichiometry was detected. The intermediate then recruits a second TonB protein. Addition of ferricrocin, a FhuA-specific ligand, enhanced amounts of the 2:1 complex but was not essential for its formation. To assess the role of the cork domain of FhuA in forming a 2:1 TonB-FhuA complex, we tested a FhuA deletion (residues 21-128) for its ability to interact with TonB. Analytical ultracentrifugation demonstrated that deletion of this region of the cork domain resulted in a 1:1 complex. Furthermore, the high-affinity 2:1 complex requires the N-terminal region of TonB. Together these in vitro experiments establish that TonB-FhuA interactions require sequential steps of kinetically limiting rearrangements. Additionally, domains that contribute to complex formation were identified in TonB and in FhuA.

  19. Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors.

    Science.gov (United States)

    Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

    2015-05-19

    In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

  20. Crystal structure of TAZ-TEAD complex reveals a distinct interaction mode from that of YAP-TEAD complex.

    Science.gov (United States)

    Kaan, Hung Yi Kristal; Chan, Siew Wee; Tan, Siew Kim Joyce; Guo, Fusheng; Lim, Chun Jye; Hong, Wanjin; Song, Haiwei

    2017-05-17

    The Hippo pathway is a tumor suppressor pathway that is implicated in the regulation of organ size. The pathway has three components: the upstream regulatory factors, the kinase core, and the downstream transcriptional machinery, which consists of YAP, TAZ (transcription co-activators) and TEAD (transcription factor). Formation of YAP/TAZ-TEAD complexes leads to the transcription of growth-promoting genes. Herein, we report the crystal structure of TAZ-TEAD4 complex, which reveals two binding modes. The first is similar to the published YAP-TEAD structure. The second is a unique binding mode, whereby two molecules of TAZ bind to and bridge two molecules of TEAD4. We validated the latter using cross-linking and multi-angle light scattering. Using siRNA, we showed that TAZ knockdown leads to a decrease in TEAD4 dimerization. Lastly, results from luciferase assays, using YAP/TAZ transfected or knockdown cells, give support to the non-redundancy of YAP/TAZ co-activators in regulating gene expression in the Hippo pathway.

  1. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    Science.gov (United States)

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  2. The organization of thinking: what functional brain imaging reveals about the neuroarchitecture of complex cognition.

    Science.gov (United States)

    Just, Marcel Adam; Varma, Sashank

    2007-09-01

    Recent findings in brain imaging, particularly in fMRI, are beginning to reveal some of the fundamental properties of the organization of the cortical systems that underpin complex cognition. We propose an emerging set of operating principles that govern this organization, characterizing the system as a set of collaborating cortical centers that operate as a large-scale cortical network. Two of the network's critical features are that it is resource constrained and dynamically configured, with resource constraints and demands dynamically shaping the network topology. The operating principles are embodied in a cognitive neuroarchitecture, 4CAPS, consisting of a number of interacting computational centers that correspond to activating cortical areas. Each 4CAPS center is a hybrid production system, possessing both symbolic and connectionist attributes. We describe 4CAPS models of sentence comprehension, spatial problem solving, and complex multitasking and compare the accounts of these models with brain activation and behavioral results. Finally, we compare 4CAPS with other proposed neuroarchitectures.

  3. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

    Directory of Open Access Journals (Sweden)

    Masakazu Kohda

    2016-01-01

    Full Text Available Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4 as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3 and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21 as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

  4. Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

    Science.gov (United States)

    Jin, Ke; Musso, Gabriel; Vlasblom, James; Jessulat, Matthew; Deineko, Viktor; Negroni, Jacopo; Mosca, Roberto; Malty, Ramy; Nguyen-Tran, Diem-Hang; Aoki, Hiroyuki; Minic, Zoran; Freywald, Tanya; Phanse, Sadhna; Xiang, Qian; Freywald, Andrew; Aloy, Patrick; Zhang, Zhaolei; Babu, Mohan

    2015-02-06

    Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

  5. Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.

    Science.gov (United States)

    Bischofberger, Mirko; Iacovache, Ioan; Boss, Daniel; Naef, Felix; van der Goot, F Gisou; Molina, Nacho

    2016-04-12

    Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.

  6. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies

    Science.gov (United States)

    Nyuzuki, Hiromi; Moriyama, Yohsuke; Mizuno, Yosuke; Hirata, Tomoko; Yatsuka, Yukiko; Yamashita-Sugahara, Yzumi; Nakachi, Yutaka; Kato, Hidemasa; Okuda, Akihiko; Tamaru, Shunsuke; Borna, Nurun Nahar; Banshoya, Kengo; Aigaki, Toshiro; Sato-Miyata, Yukiko; Ohnuma, Kohei; Suzuki, Tsutomu; Nagao, Asuteka; Maehata, Hazuki; Matsuda, Fumihiko; Higasa, Koichiro; Nagasaki, Masao; Yasuda, Jun; Yamamoto, Masayuki; Fushimi, Takuya; Shimura, Masaru; Kaiho-Ichimoto, Keiko; Harashima, Hiroko; Yamazaki, Taro; Mori, Masato; Murayama, Kei; Ohtake, Akira; Okazaki, Yasushi

    2016-01-01

    Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder. PMID:26741492

  7. Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis

    Directory of Open Access Journals (Sweden)

    Takahiro Tsujikawa

    2017-04-01

    Full Text Available Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.

  8. Complex patterns in fossilized stromatolites revealed by hyperspectral imaging (400-2496 nm).

    Science.gov (United States)

    Murphy, R J; Van Kranendonk, M J; Kelloway, S J; Wainwright, I E

    2016-09-01

    Hyperspectral imaging (400-2496 nm) was used to quantitatively map surface textures and compositional variations in stromatolites to determine whether complexity of textures could be used as evidence to support biogenicity in the absence of preserved biomarkers. Four samples of 2.72-2.4 Ga stromatolites from a variety of settings, encompassing marine and lacustrine environments, were selected for hyperspectral imaging. Images of the sawn surfaces of samples were processed to identify reflectance and mineral absorption features and quantify their intensity (as an index of mineral abundance) using automated feature extraction. Amounts of ferrous iron were quantified using a ratio of reflectance at 1650 and 1299 nm. Visible near infrared imagery (400-970 nm) did not reveal additional textural patterns to those obtained from visual inspection. Shortwave infrared imagery (1000-2496 nm), however, revealed complex laminar and convoluted patterns, including a distinctive texture of sharp peaks and broad, low troughs in one sample, similar to living tufted microbial mats. Spectral analysis revealed another sample to be composed of dolomite. Two other samples were dominated by calcite or chlorite ± illite. Large variations in amounts of ferrous iron were found, but ferric iron was exclusively located in the oxidation crust. Hyperspectral imaging revealed large differences between parts of a sample of biogenic and non-biogenic origin. The former was characterized by calcite with varying amounts of ferrous iron, distributed in lenticular, convoluted patterns; the latter by Mg-Fe chlorite with large amounts of aluminium silicate, distributed as fine laminar layers. All minerals identified by hyperspectral imaging were confirmed by thin section petrography and XRD analyses. Spatial statistics generated from quantitative minerals maps showed different patterns between these different parts of the sample. Thus, hyperspectral imaging was shown to be a powerful tool for

  9. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Structure of a Blm10 Complex Reveals Common Mechanisms for Proteasome Binding and Gate Opening

    Energy Technology Data Exchange (ETDEWEB)

    Sadre-Bazzaz, K.; Robinson, H.; Whitby, F. G.; Formosa, T.; Hill, C. P.

    2010-03-12

    The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 {angstrom} resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

  11. Single molecule atomic force microscopy of aerolysin pore complexes reveals unexpected star-shaped topography.

    Science.gov (United States)

    He, Jianfeng; Wang, Jiabin; Hu, Jun; Sun, Jielin; Czajkowsky, Daniel Mark; Shao, Zhifeng

    2016-04-01

    Aerolysin is the paradigmatic member of a large family of toxins that convert from a water-soluble monomer/dimer into a membrane-spanning oligomeric pore. While there is x-ray crystallographic data of its water-soluble conformation, the most recent structural model of the membrane-inserted pore is based primarily on data of water-soluble tetradecamers of mutant protein, together with computational modeling ultimately performed in vacuum. Here we examine this pore model with atomic force microscopy (AFM) of membrane-associated wild-type complexes and all-atom molecular dynamics (MD) simulations in water. In striking contrast to a disc-shaped cap region predicted by the present model, the AFM images reveal a star-shaped complex, with a central ring surrounded by seven radial projections. Further, the MD simulations suggest that the locations of the receptor-binding (D1) domains in the present model are not correct. However, a modified model in which the D1 domains, rather than localized at fixed positions, adopt a wide range of configurations through fluctuations of an intervening linker is compatible with existing data. Thus our work not only demonstrates the importance of directly resolving such complexes in their native environment but also points to a dynamic receptor binding region, which may be critical for toxin assembly on the cell surface.

  12. Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

    Science.gov (United States)

    Schlau-Cohen, Gabriela S; Wang, Quan; Southall, June; Cogdell, Richard J; Moerner, W E

    2013-07-01

    Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

  13. Genetic networking of the Bemisia tabaci cryptic species complex reveals pattern of biological invasions.

    Directory of Open Access Journals (Sweden)

    Paul De Barro

    Full Text Available BACKGROUND: A challenge within the context of cryptic species is the delimitation of individual species within the complex. Statistical parsimony network analytics offers the opportunity to explore limits in situations where there are insufficient species-specific morphological characters to separate taxa. The results also enable us to explore the spread in taxa that have invaded globally. METHODOLOGY/PRINCIPAL FINDINGS: Using a 657 bp portion of mitochondrial cytochrome oxidase 1 from 352 unique haplotypes belonging to the Bemisia tabaci cryptic species complex, the analysis revealed 28 networks plus 7 unconnected individual haplotypes. Of the networks, 24 corresponded to the putative species identified using the rule set devised by Dinsdale et al. (2010. Only two species proposed in Dinsdale et al. (2010 departed substantially from the structure suggested by the analysis. The analysis of the two invasive members of the complex, Mediterranean (MED and Middle East - Asia Minor 1 (MEAM1, showed that in both cases only a small number of haplotypes represent the majority that have spread beyond the home range; one MEAM1 and three MED haplotypes account for >80% of the GenBank records. Israel is a possible source of the globally invasive MEAM1 whereas MED has two possible sources. The first is the eastern Mediterranean which has invaded only the USA, primarily Florida and to a lesser extent California. The second are western Mediterranean haplotypes that have spread to the USA, Asia and South America. The structure for MED supports two home range distributions, a Sub-Saharan range and a Mediterranean range. The MEAM1 network supports the Middle East - Asia Minor region. CONCLUSION/SIGNIFICANCE: The network analyses show a high level of congruence with the species identified in a previous phylogenetic analysis. The analysis of the two globally invasive members of the complex support the view that global invasion often involve very small portions of

  14. Multilocus sequence data reveal extensive phylogenetic species diversity within the Neurospora discreta complex.

    Science.gov (United States)

    Dettman, Jeremy R; Jacobson, David J; Taylor, John W

    2006-01-01

    Previous observations of morphological, reproductive and genetic variation have suggested that Neurospora discreta, as presently circumscribed, might represent a diverse complex of multiple species. To investigate this hypothesis we examined the phylogenetic relationships among 73 fungal strains traditionally identified as N. discreta. Strains were chosen from across the morphological, ecological and geographical ranges of the species. Sequence data were obtained from three unlinked nuclear loci, and phylogenetic species recognition was applied to the dataset using protocols that have been shown to be reliable for identifying independent lineages and delineating species of Neurospora. The results demonstrate that the present circumscription of N. discreta includes at least eight separate phylogenetic species. This research also reveals an abundance of previously unrecognized genetic diversity within the genus, characterizes the interspecific evolutionary relationships and contributes to a fuller understanding of species diversity in Neurospora.

  15. Comparative analyses of developmental transcription factor repertoires in sponges reveal unexpected complexity of the earliest animals.

    Science.gov (United States)

    Fortunato, Sofia A V; Adamski, Marcin; Adamska, Maja

    2015-12-01

    Developmental transcription factors (DTFs) control development of animals by affecting expression of target genes, some of which are transcription factors themselves. In bilaterians and cnidarians, conserved DTFs are involved in homologous processes such as gastrulation or specification of neurons. The genome of Amphimedon queenslandica, the first sponge to be sequenced, revealed that only a fraction of these conserved DTF families are present in demosponges. This finding was in line with the view that morphological complexity in the animal lineage correlates with developmental toolkit complexity. However, as the phylum Porifera is very diverse, Amphimedon's genome may not be representative of all sponges. The recently sequenced genomes of calcareous sponges Sycon ciliatum and Leucosolenia complicata allowed investigations of DTFs in a sponge lineage evolutionarily distant from demosponges. Surprisingly, the phylogenetic analyses of identified DTFs revealed striking differences between the calcareous sponges and Amphimedon. As these differences appear to be a result of independent gene loss events in the two sponge lineages, the last common ancestor of sponges had to possess a much more diverse repertoire of DTFs than extant sponges. Developmental expression of sponge homologs of genes involved in specification of the Bilaterian endomesoderm and the neurosensory cells suggests that roles of many DTFs date back to the last common ancestor of all animals. Strikingly, even DTFs displaying apparent pan-metazoan conservation of sequence and function are not immune to being lost from individual species genomes. The quest for a comprehensive picture of the developmental toolkit in the last common metazoan ancestor is thus greatly benefitting from the increasing accessibility of sequencing, allowing comparisons of multiple genomes within each phylum.

  16. The integrative taxonomic approach reveals host specific species in an encyrtid parasitoid species complex.

    Directory of Open Access Journals (Sweden)

    Douglas Chesters

    Full Text Available Integrated taxonomy uses evidence from a number of different character types to delimit species and other natural groupings. While this approach has been advocated recently, and should be of particular utility in the case of diminutive insect parasitoids, there are relatively few examples of its application in these taxa. Here, we use an integrated framework to delimit independent lineages in Encyrtus sasakii (Hymenoptera: Chalcidoidea: Encyrtidae, a parasitoid morphospecies previously considered a host generalist. Sequence variation at the DNA barcode (cytochrome c oxidase I, COI and nuclear 28S rDNA loci were compared to morphometric recordings and mating compatibility tests, among samples of this species complex collected from its four scale insect hosts, covering a broad geographic range of northern and central China. Our results reveal that Encyrtus sasakii comprises three lineages that, while sharing a similar morphology, are highly divergent at the molecular level. At the barcode locus, the median K2P molecular distance between individuals from three primary populations was found to be 11.3%, well outside the divergence usually observed between Chalcidoidea conspecifics (0.5%. Corroborative evidence that the genetic lineages represent independent species was found from mating tests, where compatibility was observed only within populations, and morphometric analysis, which found that despite apparent morphological homogeneity, populations clustered according to forewing shape. The independent lineages defined by the integrated analysis correspond to the three scale insect hosts, suggesting the presence of host specific cryptic species. The finding of hidden host specificity in this species complex demonstrates the critical role that DNA barcoding will increasingly play in revealing hidden biodiversity in taxa that present difficulties for traditional taxonomic approaches.

  17. Connected magma plumbing system between Cerro Negro and El Hoyo Complex, Nicaragua revealed by gravity survey

    Science.gov (United States)

    MacQueen, Patricia; Zurek, Jeffrey; Williams-Jones, Glyn

    2016-11-01

    Cerro Negro, near León, Nicaragua is a young, relatively small basaltic cinder cone volcano that has been unusually active during its short lifespan. Multiple explosive eruptions have deposited significant amounts of ash on León and the surrounding rural communities. While a number of studies investigate the geochemistry and stress regime of the volcano, subsurface structures have only been studied by diffuse soil gas surveys. These studies have raised several questions as to the proper classification of Cerro Negro and its relation to neighboring volcanic features. To address these questions, we collected 119 gravity measurements around Cerro Negro volcano in an attempt to delineate deep structures at the volcano. The resulting complete Bouguer anomaly map revealed local positive gravity anomalies (wavelength 0.5 to 2 km, magnitude +4 mGal) and regional positive (10 km wavelength, magnitudes +10 and +8 mGal) and negative (12 and 6 km wavelength, magnitudes -18 and -13 mGal) Bouguer anomalies. Further analysis of these gravity data through inversion has revealed both local and regional density anomalies that we interpret as intrusive complexes at Cerro Negro and in the Nicaraguan Volcanic Arc. The local density anomalies at Cerro Negro have a density of 2700 kg m-3 (basalt) and are located between -250 and -2000 m above sea level. The distribution of recovered density anomalies suggests that eruptions at Cerro Negro may be tapping an interconnected magma plumbing system beneath El Hoyo, Cerro La Mula, and Cerro Negro, and more than seven other proximal volcanic features, implying that Cerro Negro should be considered the newest cone of a Cerro Negro-El Hoyo volcanic complex.

  18. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  19. Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy

    Science.gov (United States)

    Ferretti, Marco; Hendrikx, Ruud; Romero, Elisabet; Southall, June; Cogdell, Richard J.; Novoderezhkin, Vladimir I.; Scholes, Gregory D.; van Grondelle, Rienk

    2016-02-01

    Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines the selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. We suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy.

  20. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  1. Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy.

    Science.gov (United States)

    Ferretti, Marco; Hendrikx, Ruud; Romero, Elisabet; Southall, June; Cogdell, Richard J; Novoderezhkin, Vladimir I; Scholes, Gregory D; van Grondelle, Rienk

    2016-02-09

    Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines the selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. We suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy.

  2. Structure of the Sulphiredoxin-Peroxiredoxin Complex Reveals an Essential Repair Embrace

    Energy Technology Data Exchange (ETDEWEB)

    Jonsson,T.; Johnson, L.; Lowther, W.

    2008-01-01

    Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling1. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal2. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 Angstroms crystal structure of the human Srx-PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.

  3. Cryptic biodiversity effects: importance of functional redundancy revealed through addition of food web complexity.

    Science.gov (United States)

    Philpott, Stacy M; Pardee, Gabriella L; Gonthier, David J

    2012-05-01

    Interactions between predators and the degree of functional redundancy among multiple predator species may determine whether herbivores experience increased or decreased predation risk. Specialist parasites can modify predator behavior, yet rarely have cascading effects on multiple predator species and prey been evaluated. We examined influences of specialist phorid parasites (Pseudacteon spp.) on three predatory ant species and herbivores in a coffee agroecosystem. Specifically, we examined whether changes in ant richness affected fruit damage by the coffee berry borer (Hypothenemus hampei) and whether phorids altered multi-predator effects. Each ant species reduced borer damage, and without phorids, increasing predator richness did not further decrease borer damage. However, with phorids, activity of one ant species was reduced, indicating that the presence of multiple ant species was necessary to limit borer damage. In addition, phorid presence revealed synergistic effects of multiple ant species, not observed without the presence of this parasite. Thus, a trait-mediated cascade resulting from a parasite-induced predator behavioral change revealed the importance of functional redundancy, predator diversity, and food web complexity for control of this important pest.

  4. Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex.

    Science.gov (United States)

    Reboul, Cyril F; Meyer, Grischa R; Porebski, Benjamin T; Borg, Natalie A; Buckle, Ashley M

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

  5. Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex.

    Directory of Open Access Journals (Sweden)

    Cyril F Reboul

    Full Text Available The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156 located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

  6. Revealing the hidden networks of interaction in mobile animal groups allows prediction of complex behavioral contagion.

    Science.gov (United States)

    Rosenthal, Sara Brin; Twomey, Colin R; Hartnett, Andrew T; Wu, Hai Shan; Couzin, Iain D

    2015-04-14

    Coordination among social animals requires rapid and efficient transfer of information among individuals, which may depend crucially on the underlying structure of the communication network. Establishing the decision-making circuits and networks that give rise to individual behavior has been a central goal of neuroscience. However, the analogous problem of determining the structure of the communication network among organisms that gives rise to coordinated collective behavior, such as is exhibited by schooling fish and flocking birds, has remained almost entirely neglected. Here, we study collective evasion maneuvers, manifested through rapid waves, or cascades, of behavioral change (a ubiquitous behavior among taxa) in schooling fish (Notemigonus crysoleucas). We automatically track the positions and body postures, calculate visual fields of all individuals in schools of ∼150 fish, and determine the functional mapping between socially generated sensory input and motor response during collective evasion. We find that individuals use simple, robust measures to assess behavioral changes in neighbors, and that the resulting networks by which behavior propagates throughout groups are complex, being weighted, directed, and heterogeneous. By studying these interaction networks, we reveal the (complex, fractional) nature of social contagion and establish that individuals with relatively few, but strongly connected, neighbors are both most socially influential and most susceptible to social influence. Furthermore, we demonstrate that we can predict complex cascades of behavioral change at their moment of initiation, before they actually occur. Consequently, despite the intrinsic stochasticity of individual behavior, establishing the hidden communication networks in large self-organized groups facilitates a quantitative understanding of behavioral contagion.

  7. Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

    Energy Technology Data Exchange (ETDEWEB)

    Mikeska, Ruth [Institute of Biochemistry and Food Chemistry, University of Hamburg, c/o DESY, Notkestrasse 85, Building 22a, 22603 Hamburg (Germany); Wacker, Roland [Institute of Physiological Chemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen (Germany); Arni, Raghuvir [Department of Physics, IBILCE/UNESP, São Jose do Rio Preto, São Paul (Brazil); Singh, Tej P. [Department of Biophysics, All India Institute of Medical Sciences, New Delhi (India); Mikhailov, Albert; Gabdoulkhakov, Azat [Institute of Crystallography of Russian Academy of Sciences, Leninsky Prospect 59, 117333 Moscow (Russian Federation); Voelter, Wolfgang [Institute of Physiological Chemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen (Germany); Betzel, Christian, E-mail: betzel@unisgi1.desy.de [Institute of Biochemistry and Food Chemistry, University of Hamburg, c/o DESY, Notkestrasse 85, Building 22a, 22603 Hamburg (Germany)

    2005-01-01

    The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R{sub free} = 23.6%) and 20.9 (R{sub free} = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.

  8. Revealing the Organization of Complex Adaptive Systems through Multivariate Time Series Modeling

    Directory of Open Access Journals (Sweden)

    David G. Angeler

    2011-09-01

    Full Text Available Revealing the adaptive responses of ecological, social, and economic systems to a transforming biosphere is crucial for understanding system resilience and preventing collapse. However, testing the theory that underpins complex adaptive system organization (e.g., panarchy theory is challenging. We used multivariate time series modeling to identify scale-specific system organization and, by extension, apparent resilience mechanisms. We used a 20-year time series of invertebrates and phytoplankton from 26 Swedish lakes to test the proposition that a few key-structuring environmental variables at specific scales create discontinuities in community dynamics. Cross-scale structure was manifested in two independent species groups within both communities across lakes. The first species group showed patterns of directional temporal change, which was related to environmental variables that acted at broad spatiotemporal scales (reduced sulfate deposition, North Atlantic Oscillation. The second species group showed fluctuation patterns, which often could not be explained by environmental variables. However, when significant relationships were found, species-group trends were predicted by variables (total organic carbon, nutrients that acted at narrower spatial scales (i.e., catchment and lake. Although the sets of environmental variables that predicted the species groups differed between phytoplankton and invertebrates, the scale-specific imprints of keystone environmental variables for creating cross-scale structure were clear for both communities. Temporal trends of functional groups did not track the observed structural changes, suggesting functional stability despite structural change. Our approach allows for identifying scale-specific patterns and processes, thus providing opportunities for better characterization of complex adaptive systems organization and dynamics. This, in turn, holds potential for more accurate evaluation of resilience in

  9. Perched Lava Pond Complex on South Rift of Axial Volcano Revealed in AUV Mapping

    Science.gov (United States)

    Paduan, J. B.; Clague, D. A.; Caress, D. W.; Thomas, H. J.

    2013-12-01

    An extraordinary lava pond complex is located on Axial Volcano's distal south rift. It was discovered in EM300 multibeam bathymetry collected in 1998, and explored and sampled with ROVs Tiburon in 2005 and Doc Ricketts in 2013. It was surveyed with the MBARI Mapping AUV D. Allan B. in 2011, in a complicated mission first flying above the levees at constant depth, then skimming ~5 m over the levees at a different constant depth to survey the floors, then twice switching to constant altitude mode to map outside the ponds. The AUV navigation was adjusted using the MB-System tool mbnavadjust so that bathymetric features match in overlapping and crossing swaths. The ~1-m resolution AUV bathymetry reveals extremely rough terrain, where low-resolution EM300 data had averaged acoustic returns and obscured details of walls, floors, a breach and surrounding flows, and gives context to the ROV observations and samples. The 6 x 1.5 km pond complex has 4 large and several smaller drained ponds with rims 67 to 106 m above the floors. The combined volume before draining was 0.56 km3. The ponds overflowed to build lobate-flow levees with elongate pillows draping outer flanks, then drained, leaving lava veneer on vertical inner walls. Levee rim depths vary by only 10 m and are deeper around the southern ponds. Deep collapse-pits in the levees suggest porosity of pond walls. The eastern levee of the northeastern pond breached, draining the interconnected ponds, and fed thick, rapidly-emplaced, sheet-flows along the complex's east side. These flows travelled at least 5.5 km down-rift and have 19-33 m deep drained ponds. They extended up-rift as well, forming a 10 x 2.5 km ponded flow with level 'bathtub rings' as high as 35 m above the floor marking that flow's high-stand. Despite the breach, at least 0.066 km3 of the molten interior of the large ponds also drained back down the eruptive fissures, as the pond floors are deeper than the sill and sea floor outside the complex. Tumulus

  10. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    Science.gov (United States)

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  11. A Lesion-Mimic Syntaxin Double Mutant in Arabidopsis Reveals Novel Complexity of Pathogen Defense Signaling

    Institute of Scientific and Technical Information of China (English)

    Ziguo Zhang; Hans Thordal-Christensen; Andrea Lenk; Mats X. Andersson; Torben Gjetting; Carsten Pedersen; Mads E. Nielsen; Marl-Anne Newman; Bi-Huei Hou; Shauna C. Somerville

    2008-01-01

    The lesion-mimicArabidopsis mutant, syp121 syp122, constitutively expresses the salicylic acid (SA) signaling pathway and has low penetration resistance to powdery mildew fungi. Genetic analyses of the lesion-mimic phenotype have expanded our understanding of programmed cell death (PCD) in plants. Inactivation of SA signaling genes in syp121 syp 122 only partially rescues the lesion-mimic phenotype, indicating that additional defenses contribute to the PCD. Whole genome transcriptome analysis confirmed that SA-induced transcripts, as well as numerous other known pathogenresponse transcripts, are up-regulated after inactivation of the syntaxin genes. A suppressor mutant analysis of syp121 syp122 revealed that FMO1, ALD1, and PAD4 are important for lesion development. Mutant alleles of EDS1, NDR1, RAR1, and SGT1b also partially rescued the lesion-mimic phenotype, suggesting that mutating syntaxin genes stimulates TIR-NB-LRR and CC-NB-LRR-type resistances. The syntaxin double knockout potentiated a powdery mildewinduced HR-like response. This required functional PAD4 but not functional SA signaling. However, SA signaling potentiated the PAD4-dependent HR-like response. Analyses of quadruple mutants suggest that EDS5 and SID2 confer separate SA-independent signaling functions, and that FMO1 and ALD1 mediate SA-independent signals that are NPRl-dependent.These studies highlight the contribution of multiple pathways to defense and point to the complexity of their interactions.

  12. Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

    Science.gov (United States)

    Mirza, I Ahmad; Yachnin, Brahm J; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K; Berghuis, Albert M

    2009-07-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  13. Complex evolutionary patterns revealed by mitochondrial genomes of the domestic horse.

    Science.gov (United States)

    Ning, T; Li, J; Lin, K; Xiao, H; Wylie, S; Hua, S; Li, H; Zhang, Y-P

    2014-01-01

    The domestic horse is the most widely used and important stock and recreational animal, valued for its strength and endurance. The energy required by the domestic horse is mainly supplied by mitochondria via oxidative phosphorylation. Thus, selection may have played an essential role in the evolution of the horse mitochondria. Besides, demographic events also affect the DNA polymorphic pattern on mitochondria. To understand the evolutionary patterns of the mitochondria of the domestic horse, we used a deep sequencing approach to obtain the complete sequences of 15 mitochondrial genomes, and four mitochondrial gene sequences, ND6, ATP8, ATP6 and CYTB, collected from 509, 363, 363 and 409 domestic horses, respectively. Evidence of strong substitution rate heterogeneity was found at nonsynonymous sites across the genomes. Signatures of recent positive selection on mtDNA of domestic horse were detected. Specifically, five amino acids in the four mitochondrial genes were identified as the targets of positive selection. Coalescentbased simulations imply that recent population expansion is the most probable explanation for the matrilineal population history for domestic horse. Our findings reveal a complex pattern of non-neutral evolution of the mitochondrial genome in the domestic horses.

  14. Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor.

    Science.gov (United States)

    Nobu, Masaru K; Narihiro, Takashi; Rinke, Christian; Kamagata, Yoichi; Tringe, Susannah G; Woyke, Tanja; Liu, Wen-Tso

    2015-08-01

    Ecogenomic investigation of a methanogenic bioreactor degrading terephthalate (TA) allowed elucidation of complex synergistic networks of uncultivated microorganisms, including those from candidate phyla with no cultivated representatives. Our previous metagenomic investigation proposed that Pelotomaculum and methanogens may interact with uncultivated organisms to degrade TA; however, many members of the community remained unaddressed because of past technological limitations. In further pursuit, this study employed state-of-the-art omics tools to generate draft genomes and transcriptomes for uncultivated organisms spanning 15 phyla and reports the first genomic insight into candidate phyla Atribacteria, Hydrogenedentes and Marinimicrobia in methanogenic environments. Metabolic reconstruction revealed that these organisms perform fermentative, syntrophic and acetogenic catabolism facilitated by energy conservation revolving around H2 metabolism. Several of these organisms could degrade TA catabolism by-products (acetate, butyrate and H2) and syntrophically support Pelotomaculum. Other taxa could scavenge anabolic products (protein and lipids) presumably derived from detrital biomass produced by the TA-degrading community. The protein scavengers expressed complementary metabolic pathways indicating syntrophic and fermentative step-wise protein degradation through amino acids, branched-chain fatty acids and propionate. Thus, the uncultivated organisms may interact to form an intricate syntrophy-supported food web with Pelotomaculum and methanogens to metabolize catabolic by-products and detritus, whereby facilitating holistic TA mineralization to CO2 and CH4.

  15. Complex patterns of faulting revealed by 3D seismic data at the West Galicia rifted margin

    Science.gov (United States)

    Reston, Timothy; Cresswell, Derren; Sawyer, Dale; Ranero, Cesar; Shillington, Donna; Morgan, Julia; Lymer, Gael

    2015-04-01

    The west Galicia margin is characterised by crust thinning to less than 3 km, well-defined fault blocks, which overlie a bright reflection (the S reflector) generally interpreted as a tectonic Moho. The margin exhibits neither voluminous magmatism nor thick sediment piles to obscure the structures and the amount of extension. As such is represents an ideal location to study the process of continental breakup both through seismic imaging and potentially through drilling. Prestack depth migration of existing 2D profiles has strongly supported the interpretation of the S reflector as both a detachment and as the crust-mantle boundary; wide-angle seismic has also shown that the mantle beneath S is serpentinised. Despite the quality of the existing 2D seismic images, a number of competing models have been advanced to explain the formation of this margin, including sequential faulting, polyphase faulting, multiple detachments and the gravitational collapse of the margin over exhumed mantle. As these models, all developed for the Galicia margin, have been subsequently applied to other margins, distinguishing between them has implications not only for the structure of the Galicia margin but for the process of rifting through to breakup more generally. To address these issues in summer of 2013 we collected a 3D combined seismic reflection and wide-angle dataset over this margin. Here we present some of the results of ongoing processing of the 3D volume, focussing on the internal structure of some of the fault blocks that overlies the S detachment. 2D processing of the data shows a relatively simple series of tilted fault block, bound by west-dipping faults that detach downwards onto the bright S reflector. However, inspection of the 3D volume produced by 3D pre-stack time migration reveals that the fault blocks contain a complex set of sedimentary packages, with strata tilted to the east, west, north and south, each package bound by faults. Furthermore, the top of crustal

  16. Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

    Directory of Open Access Journals (Sweden)

    Chistoserdov Andrei

    2009-11-01

    Full Text Available Abstract Background Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species' of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela. Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets. Results The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA gene sequences for taxonomic

  17. Complexity of free energy landscapes of peptides revealed by nonlinear principal component analysis.

    Science.gov (United States)

    Nguyen, Phuong H

    2006-12-01

    Employing the recently developed hierarchical nonlinear principal component analysis (NLPCA) method of Saegusa et al. (Neurocomputing 2004;61:57-70 and IEICE Trans Inf Syst 2005;E88-D:2242-2248), the complexities of the free energy landscapes of several peptides, including triglycine, hexaalanine, and the C-terminal beta-hairpin of protein G, were studied. First, the performance of this NLPCA method was compared with the standard linear principal component analysis (PCA). In particular, we compared two methods according to (1) the ability of the dimensionality reduction and (2) the efficient representation of peptide conformations in low-dimensional spaces spanned by the first few principal components. The study revealed that NLPCA reduces the dimensionality of the considered systems much better, than did PCA. For example, in order to get the similar error, which is due to representation of the original data of beta-hairpin in low dimensional space, one needs 4 and 21 principal components of NLPCA and PCA, respectively. Second, by representing the free energy landscapes of the considered systems as a function of the first two principal components obtained from PCA, we obtained the relatively well-structured free energy landscapes. In contrast, the free energy landscapes of NLPCA are much more complicated, exhibiting many states which are hidden in the PCA maps, especially in the unfolded regions. Furthermore, the study also showed that many states in the PCA maps are mixed up by several peptide conformations, while those of the NLPCA maps are more pure. This finding suggests that the NLPCA should be used to capture the essential features of the systems.

  18. Correcting for Differential Transcript Coverage Reveals a Strong Relationship between Alternative Splicing and Organism Complexity

    OpenAIRE

    Chen, Lu; Bush, Stephen J; Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Urrutia, Araxi O.

    2014-01-01

    What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorti...

  19. Transcriptome profiling of taproot reveals complex regulatory networks during taproot thickening in radish (Raphanus sativus L.

    Directory of Open Access Journals (Sweden)

    Rugang Yu

    2016-08-01

    Full Text Available Radish (Raphanus sativus L., is one of the most important vegetable crops worldwide. Taproot thickening represents a critical developmental period that determines yield and quality in radish life cycle. To isolate differentially expressed genes (DGEs involved in radish taproot thickening process and explored the molecular mechanism in underlying taproot development, three cDNA libraries from radish taproot collected at pre-cortex splitting stage (L1, cortex splitting stage (L2 and expanding stage (L3 were constructed and sequenced by RNA-Seq technology. More than seven million clean reads were obtained from the three libraries, respectively, from which 4,717,617 (L1, 65.35%, 4,809,588 (L2, 68.24% and 4,973,745 (L3, 69.45% reads were matched to the radish reference genes. A total of 85,939 transcripts were generated from three libraries, from which 10,450, 12,325 and 7,392 differentially expressed transcripts (DETs were detected in L1 vs. L2, L1 vs. L3, and L2 vs. L3 comparisons, respectively. Gene Ontology and pathway analysis showed that many DEGs, including EXPA9, Cyclin, CaM, Syntaxin, MADS-box, SAUR and CalS were involved in cell events, cell wall modification, regulation of plant hormone levels, signal transduction and metabolisms, which may relate to taproot thickening. Furthermore, the integrated analysis of mRNA-miRNA revealed that 43 miRNAs and 92 genes that formed 114 miRNA-target mRNA pairs were co-expressed, and three miRNA-target regulatory networks of taproot were constructed from different libraries. Finally, the expression patterns of 16 selected genes were confirmed using RT-qPCR analysis. A hypothetical model of genetic regulatory network associated with taproot thickening in radish was put forward. The taproot formation of radish is mainly contributed to cell differentiation, division and expansion, which are regulated and promoted by certain specific signal transduction pathways and metabolism possesses. These results could

  20. Cryo-EM structure of respiratory complex I reveals a link to mitochondrial sulfur metabolism.

    Science.gov (United States)

    D'Imprima, Edoardo; Mills, Deryck J; Parey, Kristian; Brandt, Ulrich; Kühlbrandt, Werner; Zickermann, Volker; Vonck, Janet

    2016-12-01

    Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating H2S from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.

  1. Comprehensive genetic analysis of OEIS complex reveals no evidence for a recurrent microdeletion or duplication

    NARCIS (Netherlands)

    Vlangos, C.N.; Siuniak, A.; Ackley, T.; Bokhoven, H. van; Veltman, J.A.; Iyer, R.; Park, J.M.; Keppler-Noreuil, K.; Keegan, C.E.

    2011-01-01

    Omphalocele-exstrophy of the bladder-imperforate anus-spinal defects (OEIS) complex, or cloacal exstrophy (EC), is a rare constellation of malformations in humans involving the urogenital, gastrointestinal, and skeletal systems, and less commonly the central nervous system. Although OEIS complex is

  2. Modularity in protein complex and drug interactions reveals new polypharmacological properties.

    Directory of Open Access Journals (Sweden)

    Jose C Nacher

    Full Text Available Recent studies have highlighted the importance of interconnectivity in a large range of molecular and human disease-related systems. Network medicine has emerged as a new paradigm to deal with complex diseases. Connections between protein complexes and key diseases have been suggested for decades. However, it was not until recently that protein complexes were identified and classified in sufficient amounts to carry out a large-scale analysis of the human protein complex system. We here present the first systematic and comprehensive set of relationships between protein complexes and associated drugs and analyzed their topological features. The network structure is characterized by a high modularity, both in the bipartite graph and in its projections, indicating that its topology is highly distinct from a random network and that it contains a rich and heterogeneous internal modular structure. To unravel the relationships between modules of protein complexes, drugs and diseases, we investigated in depth the origins of this modular structure in examples of particular diseases. This analysis unveils new associations between diseases and protein complexes and highlights the potential role of polypharmacological drugs, which target multiple cellular functions to combat complex diseases driven by gain-of-function mutations.

  3. Polymorphism of DNA-anionic liposome complexes reveals hierarchy of ion-mediated interactions.

    Science.gov (United States)

    Liang, Hongjun; Harries, Daniel; Wong, Gerard C L

    2005-08-09

    Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid-DNA complexes because of their low cytotoxicity. We investigate AL-DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the self-assembled structures and phase behavior of AL-DNA complexes. The governing interactions in AL-DNA systems are complex: divalent ions can mediate strong attractions between different combinations of the components (such as DNA-DNA and membrane-membrane). Moreover, divalent cations can coordinate non-electrostatically with lipids and modify the resultant membrane structure. We find that at low membrane charge densities AL-DNA complexes organize into a lamellar structure of alternating DNA and membrane layers crosslinked by ions. At high membrane charge densities, a new phase with no analog in cationic lipid-DNA systems is observed: DNA is expelled from the complex, and a lamellar stack of membranes and intercalated ions is formed. For a subset of the ionic species, high ion concentrations generate an inverted hexagonal phase comprised of DNA strands wrapped by ion-coated lipid tubes. A simple theoretical model that takes into account the electrostatic and membrane elastic contributions to the free energy shows that this transition is consistent with an ion-induced change in the membrane spontaneous curvature, c0. Moreover, the crossover between the lamellar and inverted hexagonal phases occurs at a critical c0 that agrees well with experimental values.

  4. Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival.

    Science.gov (United States)

    Smithson, Laura J; Gutmann, David H

    2016-06-15

    As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Herein, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for mTOR-mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types.

  5. Survey of large protein complexes D. vulgaris reveals great structural diversity

    Energy Technology Data Exchange (ETDEWEB)

    Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

    2009-08-15

    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

  6. Genome-wide association data reveal a global map of genetic interactions among protein complexes.

    Directory of Open Access Journals (Sweden)

    Gregory Hannum

    2009-12-01

    Full Text Available This work demonstrates how gene association studies can be analyzed to map a global landscape of genetic interactions among protein complexes and pathways. Despite the immense potential of gene association studies, they have been challenging to analyze because most traits are complex, involving the combined effect of mutations at many different genes. Due to lack of statistical power, only the strongest single markers are typically identified. Here, we present an integrative approach that greatly increases power through marker clustering and projection of marker interactions within and across protein complexes. Applied to a recent gene association study in yeast, this approach identifies 2,023 genetic interactions which map to 208 functional interactions among protein complexes. We show that such interactions are analogous to interactions derived through reverse genetic screens and that they provide coverage in areas not yet tested by reverse genetic analysis. This work has the potential to transform gene association studies, by elevating the analysis from the level of individual markers to global maps of genetic interactions. As proof of principle, we use synthetic genetic screens to confirm numerous novel genetic interactions for the INO80 chromatin remodeling complex.

  7. Transcranial magnetic stimulation reveals complex cognitive control representations in the rostral frontal cortex.

    Science.gov (United States)

    Bahlmann, J; Beckmann, I; Kuhlemann, I; Schweikard, A; Münte, T F

    2015-08-06

    Convergent evidence suggests that the lateral frontal cortex is at the heart of a brain network subserving cognitive control. Recent theories assume a functional segregation along the rostro-caudal axis of the lateral frontal cortex based on differences in the degree of complexity of cognitive control. However, the functional contribution of specific rostral and caudal sub-regions remains elusive. Here we investigate the impact of disrupting rostral and caudal target regions on cognitive control processes, using Transcranial Magnetic Stimulation (TMS). Participants performed three different task-switching conditions that assessed differences in the degree of complexity of cognitive control processes, after temporally disrupting rostral, or caudal target regions, or a control region. Disrupting the rostral lateral frontal region specifically impaired behavioral performance of the most complex task-switching condition, in comparison to the caudal target region and the control region. These novel findings shed light on the neuroanatomical architecture supporting control over goal-directed behavior.

  8. Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation

    DEFF Research Database (Denmark)

    Czaban, Adrian; Sharma, Sapna; Byrne, Stephen

    2015-01-01

    -Festuca complex show very diverse phenotypes, including for many agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity. Results We have generated de novo transcriptome assemblies for four...... clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity...

  9. Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex

    NARCIS (Netherlands)

    Cabral, A.; Groenewald, J.Z.; Rego, C.; Oliveira, H.; Crous, P.W.

    2012-01-01

    Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and

  10. Peeling Back the Layers of Policy and School Reform: Revealing the Structural and Social Complexities within

    Science.gov (United States)

    Woodside-Jiron, Haley; Gehsmann, Kristin M.

    2009-01-01

    This article explores the complex process of school change over a six-year period in one high-poverty, urban elementary school in a northeastern city of the United States. The school included in this instrumental case study was identified by its State Department of Education as "being in need of improvement" in March 2000. Findings…

  11. Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

    NARCIS (Netherlands)

    Taylor, D.W.; Zhu, Y.; Staals, R.H.J.; Kornfeld, J.E.; Shinkai, A.; Oost, van der J.; Nogales, E.; Doudna, J.A.

    2015-01-01

    Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas

  12. How Can We Explain Poverty? Case Study of Dee Reveals the Complexities

    Science.gov (United States)

    Seccombe, Karen

    2011-01-01

    Many theories have been offered to explain why people are impoverished. This article by Karen Seccombe uses the case study of "Dee," a newly single mother, to explore four of the most common: individualism, social structuralism, the culture of poverty, and fatalism. She concludes that poverty is a highly complex phenomenon, and it is likely that…

  13. Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex

    NARCIS (Netherlands)

    Cabral, A.; Groenewald, J.Z.; Rego, C.; Oliveira, H.; Crous, P.W.

    2012-01-01

    Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and

  14. Proteomic analysis of exported chaperone/co-chaperone complexes of P. falciparum reveals an array of complex protein-protein interactions

    Science.gov (United States)

    Zhang, Qi; Ma, Cheng; Oberli, Alexander; Zinz, Astrid; Engels, Sonja; Przyborski, Jude M.

    2017-01-01

    Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure. To address this, we have utilized chemical cross-linking followed by mass spectrometry and immunoblotting to isolate and characterize proteins complexes containing an exported Hsp40 (PFE55), and the only known exported Hsp70 (PfHsp70x). Our data reveal that both of these proteins are contained in high molecular weight protein complexes. These complexes are found both in the infected erythrocyte, and within the parasite-derived compartment referred to as the parasitophorous vacuole. Surprisingly, our data also reveal an association of PfHsp70x with components of PTEX, a putative protein translocon within the membrane of the parasitophorous vacuole. Our results suggest that the P. falciparum- infected human erythrocyte contains numerous high molecular weight protein complexes, which may potentially be involved in host cell modification. PMID:28218284

  15. The analysis of eight transcriptomes from all poriferan classes reveals surprising genetic complexity in sponges.

    Science.gov (United States)

    Riesgo, Ana; Farrar, Nathan; Windsor, Pamela J; Giribet, Gonzalo; Leys, Sally P

    2014-05-01

    Sponges (Porifera) are among the earliest evolving metazoans. Their filter-feeding body plan based on choanocyte chambers organized into a complex aquiferous system is so unique among metazoans that it either reflects an early divergence from other animals prior to the evolution of features such as muscles and nerves, or that sponges lost these characters. Analyses of the Amphimedon and Oscarella genomes support this view of uniqueness-many key metazoan genes are absent in these sponges-but whether this is generally true of other sponges remains unknown. We studied the transcriptomes of eight sponge species in four classes (Hexactinellida, Demospongiae, Homoscleromorpha, and Calcarea) specifically seeking genes and pathways considered to be involved in animal complexity. For reference, we also sought these genes in transcriptomes and genomes of three unicellular opisthokonts, two sponges (A. queenslandica and O. carmela), and two bilaterian taxa. Our analyses showed that all sponge classes share an unexpectedly large complement of genes with other metazoans. Interestingly, hexactinellid, calcareous, and homoscleromorph sponges share more genes with bilaterians than with nonbilaterian metazoans. We were surprised to find representatives of most molecules involved in cell-cell communication, signaling, complex epithelia, immune recognition, and germ-lineage/sex, with only a few, but potentially key, absences. A noteworthy finding was that some important genes were absent from all demosponges (transcriptomes and the Amphimedon genome), which might reflect divergence from main-stem lineages including hexactinellids, calcareous sponges, and homoscleromorphs. Our results suggest that genetic complexity arose early in evolution as shown by the presence of these genes in most of the animal lineages, which suggests sponges either possess cryptic physiological and morphological complexity and/or have lost ancestral cell types or physiological processes.

  16. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  17. Unusual anal fin in a Devonian jawless vertebrate reveals complex origins of paired appendages.

    Science.gov (United States)

    Sansom, Robert S; Gabbott, Sarah E; Purnell, M A

    2013-06-23

    Jawed vertebrates (gnathostomes) have undergone radical anatomical and developmental changes in comparison with their jawless cousins (cyclostomes). Key among these is paired appendages (fins, legs and wings), which first evolved at some point on the gnathostome stem. The anatomy of fossil stem gnathostomes is, therefore, fundamental to our understanding of the nature and timing of the origin of this complex innovation. Here, we show that Euphanerops, a fossil jawless fish from the Devonian, possessed paired anal-fin radials, but no pectoral or pelvic fins. This unique condition occurs at an early stage on the stem-gnathostome lineage. This condition, and comparison with the varied condition of paired fins in other ostracoderms, indicates that there was a large amount of developmental plasticity during this episode-rather than a gradual evolution of this complex feature. Apparently, a number of different clades were exploring morphospace or undergoing multiple losses.

  18. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  19. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes.

    Science.gov (United States)

    Li, Y; Zakharov, D; Zhao, S; Tappero, R; Jung, U; Elsen, A; Baumann, Ph; Nuzzo, R G; Stach, E A; Frenkel, A I

    2015-06-29

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction-ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  20. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    Science.gov (United States)

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction--ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  1. Landscape of histone modifications in a sponge reveals the origin of animal cis-regulatory complexity

    Science.gov (United States)

    Gaiti, Federico; Jindrich, Katia; Fernandez-Valverde, Selene L; Roper, Kathrein E; Degnan, Bernard M; Tanurdžić, Miloš

    2017-01-01

    Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear when this regulatory system evolved. By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedonqueenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity. DOI: http://dx.doi.org/10.7554/eLife.22194.001 PMID:28395144

  2. Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown.

    Science.gov (United States)

    Jain, Ankur; Arauz, Edwin; Aggarwal, Vasudha; Ikon, Nikita; Chen, Jie; Ha, Taekjip

    2014-12-16

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential.

  3. A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

    Science.gov (United States)

    Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

    2016-02-01

    During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

  4. A Hidden State in Light-Harvesting Complex II Revealed By Multipulse Spectroscopy

    OpenAIRE

    Oort, Bart van; van Grondelle, Rienk; van Stokkum, Ivo H. M.

    2015-01-01

    Light-harvesting complex II (LHCII) is pivotal both for collecting solar radiation for photosynthesis, and for protection against photodamage under high light intensities (via a process called nonphotochemical quenching, NPQ). Aggregation of LHCII is associated with fluorescence quenching, and is used as an in vitro model system of NPQ. However, there is no agreement on the nature of the quencher and on the validity of aggregation as a model system. Here, we use ultrafast multipulse spectrosc...

  5. Genome-wide analysis reveals a complex pattern of genomic imprinting in mice.

    Directory of Open Access Journals (Sweden)

    Jason B Wolf

    2008-06-01

    Full Text Available Parent-of-origin-dependent gene expression resulting from genomic imprinting plays an important role in modulating complex traits ranging from developmental processes to cognitive abilities and associated disorders. However, while gene-targeting techniques have allowed for the identification of imprinted loci, very little is known about the contribution of imprinting to quantitative variation in complex traits. Most studies, furthermore, assume a simple pattern of imprinting, resulting in either paternal or maternal gene expression; yet, more complex patterns of effects also exist. As a result, the distribution and number of different imprinting patterns across the genome remain largely unexplored. We address these unresolved issues using a genome-wide scan for imprinted quantitative trait loci (iQTL affecting body weight and growth in mice using a novel three-generation design. We identified ten iQTL that display much more complex and diverse effect patterns than previously assumed, including four loci with effects similar to the callipyge mutation found in sheep. Three loci display a new phenotypic pattern that we refer to as bipolar dominance, where the two heterozygotes are different from each other while the two homozygotes are identical to each other. Our study furthermore detected a paternally expressed iQTL on Chromosome 7 in a region containing a known imprinting cluster with many paternally expressed genes. Surprisingly, the effects of the iQTL were mostly restricted to traits expressed after weaning. Our results imply that the quantitative effects of an imprinted allele at a locus depend both on its parent of origin and the allele it is paired with. Our findings also show that the imprinting pattern of a locus can be variable over ontogenetic time and, in contrast to current views, may often be stronger at later stages in life.

  6. Proteomic analyses reveal distinct chromatin-associated and soluble transcription factor complexes.

    Science.gov (United States)

    Li, Xu; Wang, Wenqi; Wang, Jiadong; Malovannaya, Anna; Xi, Yuanxin; Li, Wei; Guerra, Rudy; Hawke, David H; Qin, Jun; Chen, Junjie

    2015-01-21

    The current knowledge on how transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated by protein-protein interactions remains limited. Here, we performed proteomics analyses of soluble and chromatin-associated complexes of 56 TFs, including the targets of many signalling pathways involved in development and cancer, and 37 members of the Forkhead box (FOX) TF family. Using tandem affinity purification followed by mass spectrometry (TAP/MS), we performed 214 purifications and identified 2,156 high-confident protein-protein interactions. We found that most TFs form very distinct protein complexes on and off chromatin. Using this data set, we categorized the transcription-related or unrelated regulators for general or specific TFs. Our study offers a valuable resource of protein-protein interaction networks for a large number of TFs and underscores the general principle that TFs form distinct location-specific protein complexes that are associated with the different regulation and diverse functions of these TFs.

  7. Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

    Science.gov (United States)

    Rosenblum, Erica Bree; James, Timothy Y; Zamudio, Kelly R; Poorten, Thomas J; Ilut, Dan; Rodriguez, David; Eastman, Jonathan M; Richards-Hrdlicka, Katy; Joneson, Suzanne; Jenkinson, Thomas S; Longcore, Joyce E; Parra Olea, Gabriela; Toledo, Luís Felipe; Arellano, Maria Luz; Medina, Edgar M; Restrepo, Silvia; Flechas, Sandra Victoria; Berger, Lee; Briggs, Cheryl J; Stajich, Jason E

    2013-06-01

    Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

  8. CrypticspeciescompositionandgeneticdiversitywithinBemisiatabaci complex in soybean in India revealed by mtCOI DNA sequence

    Institute of Scientific and Technical Information of China (English)

    Prasanna H C; Kanakala S; Archana K; Jyothsna P; Varma R K; Malathi V G

    2015-01-01

    Bemisia tabaci is a cryptic species complex, causing signiifcant loss on many agricultural y important crops worldwide. Knowledge on species composition and diversity within B. tabaci complex is critical for evolving sustainable pest management strategies. Here we investigate the whitelfy species complex in soybean in major soybean growing states of India. The mitochondrial cytochrome oxidase gene subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian methods indicated the existence of three cryptic species namely Asia I, Asia II 1, and Asia II 7. Al the haplotypes detected in the study could be assigned to these three cryptic species fol owing the species demarcation criteria of 3.5%divergence threshold. Of these, Asia II 1 was found to be predominant with wide spread distribution across the surveyed regions from cool temperate zones to hot and humid tropical plains. On the contrary, cryptic species Asia II 7 showed localized distribu-tion. The Asia II 1 exhibited the highest haplotype diversity and Asia I showed high level of nucleotide diversity. There was a signiifcantly high genetic differentiation among these three cryptic species. The MEAM 1, a dreadful invasive species was not detected in the specimens tested in the current study. The diversity and distribution of three cryptic species is discussed in the light of current knowledge on distribution of whitelfy species in India and yel ow mosaic disease observed during sampling survey.

  9. Metagenomic signatures of a tropical mining-impacted stream reveal complex microbial and metabolic networks.

    Science.gov (United States)

    Reis, Mariana P; Dias, Marcela F; Costa, Patrícia S; Ávila, Marcelo P; Leite, Laura R; de Araújo, Flávio M G; Salim, Anna C M; Bucciarelli-Rodriguez, Mônica; Oliveira, Guilherme; Chartone-Souza, Edmar; Nascimento, Andréa M A

    2016-10-01

    Bacteria from aquatic ecosystems significantly contribute to biogeochemical cycles, but details of their community structure in tropical mining-impacted environments remain unexplored. In this study, we analyzed a bacterial community from circumneutral-pH tropical stream sediment by 16S rRNA and shotgun deep sequencing. Carrapatos stream sediment, which has been exposed to metal stress due to gold and iron mining (21 [g Fe]/kg), revealed a diverse community, with predominance of Proteobacteria (39.4%), Bacteroidetes (12.2%), and Parcubacteria (11.4%). Among Proteobacteria, the most abundant reads were assigned to neutrophilic iron-oxidizing taxa, such as Gallionella, Sideroxydans, and Mariprofundus, which are involved in Fe cycling and harbor several metal resistance genes. Functional analysis revealed a large number of genes participating in nitrogen and methane metabolic pathways despite the low concentrations of inorganic nitrogen in the Carrapatos stream. Our findings provide important insights into bacterial community interactions in a mining-impacted environment.

  10. The Precambrian Singo Igneous Complex (SIC), Uganda Revealed As a Mineralized Nested Ring Complex Using High Resolution Airborne Radiometric and Magnetic Data.

    Science.gov (United States)

    Atekwana, E. A.; LePera, A.; Abdelsalam, M. G.; Katumwehe, A. B.; Achang, M.

    2014-12-01

    We used high-resolution radiometrics and aeromagnetic data to investigate the Precambrian Singo Igneous Complex (SIC) in Uganda. The SIC covers an area of about 700 km² and is host to hydrothermally formed economic minerals such as Gold and Tungsten. The distribution of the ore deposits is not well known and current mine workings are limited to the western margins of the complex. Our objectives were to (1) provide a detailed geological map of the SIC and surrounding, (2) investigate relationships between preserved intrusive bodies and Precambrian tectonic structures to provide insight into emplacement of the complex, (3) examine links between magma emplacement, discontinuities and hydrothermal alteration (4) generate two-dimensional (2-D) and three-dimensional (3-D) models of the complex to understand its subsurface geometry, (5) investigate the relationship between the structure of the SIC and mineral occurrences as an aid to future exploration programs. Edge enhancement filters such as the analytical signal, vertical and tilt derivatives were applied to the magnetic data. In addition, 2-D and 3-D models were produced using Geosoft's GM-SYS 2-D and Voxi modules. The filtered data provided unprecedented structural details of the complex and revealed the following: (1) the edge of the SIC is characterized by higher magnetic susceptibility and Thorium content than its interior, (2) the SIC is characterized by eight to nine nested ring complexes with diameters ranging from 2.5 to 14 km, (3) the 3-D inversion suggests near vertical walls for the ring complexes extending to a depth of about 7 km, (4) the SIC was emplaced within a Precambrian folded basement and was traversed by numerous NW-trending dykes and (5) present day mining activities are concentrated within the folded basement units although occurrences of Tungsten and Gold are found associated with the highly magnetized edge of the ring complexes. We interpret the highly magnetized edges of the nested ring

  11. Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator.

    Science.gov (United States)

    Imada, Katsumi; Minamino, Tohru; Uchida, Yumiko; Kinoshita, Miki; Namba, Keiichi

    2016-03-29

    FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3β3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2 shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2-FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.

  12. Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches.

    Directory of Open Access Journals (Sweden)

    Ghia M Euskirchen

    2011-03-01

    Full Text Available A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5' ends, RNA Polymerases II and III, and enhancers as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins. Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of

  13. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

    DEFF Research Database (Denmark)

    Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

    2011-01-01

    Complement acts as a danger-sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non-catalytic substrate contacting subunit (C3b or C4b) in complex...... for substrate recognition by the convertases is presented based on the C5-CVF and C3b-Bb-SCIN structures. Prior knowledge concerning interactions between the endogenous convertases and their substrates is rationalized by this model....

  14. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    OpenAIRE

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unrav...

  15. Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

    DEFF Research Database (Denmark)

    Zhang, Guojie; Guo, Guangwu; Hu, Xueda

    2010-01-01

    present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative...... fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell...

  16. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Science.gov (United States)

    Munir, Riffat I; Schellenberg, John; Henrissat, Bernard; Verbeke, Tobin J; Sparling, Richard; Levin, David B

    2014-01-01

    Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  17. Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition

    Science.gov (United States)

    Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl

    2016-01-01

    Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154

  18. RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.

    Directory of Open Access Journals (Sweden)

    Isabelle Derré

    2007-10-01

    Full Text Available Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.

  19. Chemical Diversity and Complexity of Scotch Whisky as Revealed by High-Resolution Mass Spectrometry

    Science.gov (United States)

    Kew, Will; Goodall, Ian; Clarke, David; Uhrín, Dušan

    2017-01-01

    Scotch Whisky is an important product, both culturally and economically. Chemically, Scotch Whisky is a complex mixture, which comprises thousands of compounds, the nature of which are largely unknown. Here, we present a thorough overview of the chemistry of Scotch Whisky as observed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Eighty-five whiskies, representing the majority of Scotch Whisky produced and sold, were analyzed by untargeted high-resolution mass spectrometry. Thousands of chemical formulae were assigned for each sample based on parts-per-billion mass accuracy of FT-ICR MS spectra. For the first time, isotopic fine structure analysis was used to confirm the assignment of high molecular weight CHOS species in Scotch Whisky. The assigned spectra were compared using a number of visualization techniques, including van Krevelen diagrams, double bond equivalence (DBE) plots, as well as heteroatomic compound class distributions. Additionally, multivariate analysis, including PCA and OPLS-DA, was used to interpret the data, with key compounds identified for discriminating between types of whisky (blend or malt) or maturation wood type. FT-ICR MS analysis of Scotch Whisky was shown to be of significant potential in further understanding of the complexity of mature spirit drinks and as a tool for investigating the chemistry of the maturation processes.

  20. The cometary composition of a protoplanetary disk as revealed by complex cyanides

    CERN Document Server

    Oberg, Karin I; Furuya, Kenji; Qi, Chunhua; Aikawa, Yuri; Andrews, Sean M; Loomis, Ryan; Wilner, David J

    2015-01-01

    Observations of comets and asteroids show that the Solar Nebula that spawned our planetary system was rich in water and organic molecules. Bombardment brought these organics to the young Earth's surface, seeding its early chemistry. Unlike asteroids, comets preserve a nearly pristine record of the Solar Nebula composition. The presence of cyanides in comets, including 0.01% of methyl cyanide (CH3CN) with respect to water, is of special interest because of the importance of C-N bonds for abiotic amino acid synthesis. Comet-like compositions of simple and complex volatiles are found in protostars, and can be readily explained by a combination of gas-phase chemistry to form e.g. HCN and an active ice-phase chemistry on grain surfaces that advances complexity[3]. Simple volatiles, including water and HCN, have been detected previously in Solar Nebula analogues - protoplanetary disks around young stars - indicating that they survive disk formation or are reformed in situ. It has been hitherto unclear whether the s...

  1. Molecular phylogeny of Glossodoris (Ehrenberg, 1831) nudibranchs and related genera reveals cryptic and pseudocryptic species complexes

    KAUST Repository

    Matsuda, Shayle B.

    2017-03-01

    Chromodorid nudibranchs (Chromodorididae) are brightly coloured sea slugs that live in some of the most biodiverse and threatened coral reefs on the planet. However, the evolutionary relationships within this family have not been well understood, especially in the genus Glossodoris. Members of Glossodoris have experienced large-scale taxonomic instability over the last century and have been the subject of repeated taxonomic changes, in part due to morphological characters being the sole traditional taxonomic sources of data. Changing concepts of traditional generic boundaries based on morphology also have contributed to this instability. Despite recent advances in molecular systematics, many aspects of chromodorid taxonomy remain poorly understood, particularly at the traditional species and generic levels. In this study, 77 individuals comprising 32 previously defined species were used to build the most robust phylogenetic tree of Glossodoris and related genera using mitochondrial genes cytochrome c oxidase subunit I and 16S, and the nuclear gene 28S. Bayesian inference, maximum likelihood, and maximum parsimony analyses verify the most recent hypothesized evolutionary relationships within Glossodoris. Additionally, a pseudocryptic and cryptic species complex within Glossodoris cincta and a pseudocryptic complex within Glossodoris pallida emerged, and three new species of Doriprismatica are identified.

  2. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

  3. Ubiquitin dynamics in complexes reveal molecular recognition mechanisms beyond induced fit and conformational selection.

    Directory of Open Access Journals (Sweden)

    Jan H Peters

    Full Text Available Protein-protein interactions play an important role in all biological processes. However, the principles underlying these interactions are only beginning to be understood. Ubiquitin is a small signalling protein that is covalently attached to different proteins to mark them for degradation, regulate transport and other functions. As such, it interacts with and is recognised by a multitude of other proteins. We have conducted molecular dynamics simulations of ubiquitin in complex with 11 different binding partners on a microsecond timescale and compared them with ensembles of unbound ubiquitin to investigate the principles of their interaction and determine the influence of complex formation on the dynamic properties of this protein. Along the main mode of fluctuation of ubiquitin, binding in most cases reduces the conformational space available to ubiquitin to a subspace of that covered by unbound ubiquitin. This behaviour can be well explained using the model of conformational selection. For lower amplitude collective modes, a spectrum of zero to almost complete coverage of bound by unbound ensembles was observed. The significant differences between bound and unbound structures are exclusively situated at the binding interface. Overall, the findings correspond neither to a complete conformational selection nor induced fit scenario. Instead, we introduce a model of conformational restriction, extension and shift, which describes the full range of observed effects.

  4. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Directory of Open Access Journals (Sweden)

    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  5. Starch industry wastewater for production of biopesticides--ramifications of solids concentrations.

    Science.gov (United States)

    Vu, Khanh Dang; Tyagi, R D; Brar, S K; Valéro, J R; Surampalli, R Y

    2009-04-01

    Total solids (TS) concentrations ranging from 15 to 66 g L(-1) of starch industry wastewater (SIW) were tested as raw material for the production of Bacillus thuringiensis var. kurstaki HD-1 (Btk) biopesticide in shake flasks and a 15 L bench-scale fermenter. Shake flask studies revealed a higher delta-endotoxin concentration of Btk at 30 g L(-1) TS concentration and 2.5% (v v(-1)) volume of pre-culture. The fermenter experiments conducted using SIW at 30 g L(-1) TS concentration under controlled conditions of temperature, pH and dissolved oxygen showed higher spore count, enzyme production (protease and amylase) and delta-endotoxin concentration as compared with those of SIW at 15 g L(-1) TS concentration. The entomotoxicity, at the end of fermentation, with SIW at 30 g L(-1) solids concentration (17.8 x 10(9) SBU L(-1), measured against spruce budworm) was considerably higher as compared with entomotoxicity at 15 g L(-1) solids concentration (15.3 x 10(9) SBU L(-1)) and semi-synthetic medium (11.7 x 10(9) SBU L(-1)). The pellet, comprising spores and delta-endotoxin complex obtained after centrifugation and followed by resuspension (in supernatant) in one-tenth of the original volume, of SIW at 30 g L(-1) solids concentration media registered the highest potential for application (to protect forests against spruce budworm) than other media in term of entomotoxicity.

  6. Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

    Science.gov (United States)

    Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir; Singh, Tej P.; Mikhailov, Albert; Gabdoulkhakov, Azat; Voelter, Wolfgang; Betzel, Christian

    2005-01-01

    The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R free = 23.6%) and 20.9 (R free = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound. PMID:16508080

  7. RNA-Seq reveals complex genetic response to deepwater horizon oil release in Fundulus grandis

    Directory of Open Access Journals (Sweden)

    Garcia Tzintzuni I

    2012-09-01

    Full Text Available Abstract Background The release of oil resulting from the blowout of the Deepwater Horizon (DH drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future. Results Our de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set. Conclusion RNA-Seq may be successfully applied to feral and

  8. Computer simulations reveal complex distribution of haemodynamic forces in a mouse retina model of angiogenesis

    CERN Document Server

    Bernabeu, Miguel O; Jones, Martin; Nielsen, Jens H; Krüger, Timm; Nash, Rupert W; Groen, Derek; Hetherington, James; Gerhardt, Holger; Coveney, Peter V

    2013-01-01

    There is currently limited understanding of the role played by haemodynamic forces on the processes governing vascular development. One of many obstacles to be overcome is being able to measure those forces, at the required resolution level, on vessels only a few micrometres thick. In the current paper, we present an in silico method for the computation of the haemodynamic forces experienced by murine retinal vasculature (a widely used vascular development animal model) beyond what is measurable experimentally. Our results show that it is possible to reconstruct high-resolution three-dimensional geometrical models directly from samples of retinal vasculature and that the lattice-Boltzmann algorithm can be used to obtain accurate estimates of the haemodynamics in these domains. Our findings show that the flow patterns recovered are complex, that branches of predominant flow exist from early development stages, and that the pruning process tends to make the wall shear stress experienced by the capillaries incre...

  9. Molecular data reveal complex hybridization and a cryptic species of neotropical wild cat.

    Science.gov (United States)

    Trigo, Tatiane C; Schneider, Alexsandra; de Oliveira, Tadeu G; Lehugeur, Livia M; Silveira, Leandro; Freitas, Thales R O; Eizirik, Eduardo

    2013-12-16

    Hybridization among animal species has recently become more recognized as an important phenomenon, especially in the context of recent radiations. Here we show that complex hybridization has led to contrasting patterns of genomic composition among closely related species of the Neotropical cat genus Leopardus. We show strong evidence of ancient hybridization and introgression between the pampas cat (L. colocolo) and northeastern populations of tigrina (L. tigrinus), leading to remarkable cytonuclear discordance in the latter. In contrast, southern tigrina populations show recent and continuing hybridization with Geoffroy's cat (L. geoffroyi), leading to extreme levels of interspecific admixture at their contact zone. Finally, we demonstrate that two seemingly continuous Brazilian tigrina populations show no evidence of ongoing gene flow between them, leading us to support their formal recognition as distinct species, namely L. tigrinus in the northeast and L. guttulus in the south.

  10. A Polychaete's powerful punch: venom gland transcriptomics of Glycera reveals a complex cocktail of toxin homologs.

    Science.gov (United States)

    von Reumont, Björn M; Campbell, Lahcen I; Richter, Sandy; Hering, Lars; Sykes, Dan; Hetmank, Jörg; Jenner, Ronald A; Bleidorn, Christoph

    2014-09-05

    Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Revealing the complexity of a monogenic disease: rett syndrome exome sequencing.

    Directory of Open Access Journals (Sweden)

    Elisa Grillo

    Full Text Available Rett syndrome (OMIM#312750 is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005 mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome, while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant. In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.

  12. Revealing the complexity of a monogenic disease: rett syndrome exome sequencing.

    Science.gov (United States)

    Grillo, Elisa; Lo Rizzo, Caterina; Bianciardi, Laura; Bizzarri, Veronica; Baldassarri, Margherita; Spiga, Ottavia; Furini, Simone; De Felice, Claudio; Signorini, Cinzia; Leoncini, Silvia; Pecorelli, Alessandra; Ciccoli, Lucia; Mencarelli, Maria Antonietta; Hayek, Joussef; Meloni, Ilaria; Ariani, Francesca; Mari, Francesca; Renieri, Alessandra

    2013-01-01

    Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome), while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.

  13. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    Energy Technology Data Exchange (ETDEWEB)

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada (Hiroshima); (WIS-I); (UIC)

    2010-04-26

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

  14. Scalably Revealing the Dynamics of Soft Community Structure in Complex Networks

    Institute of Scientific and Technical Information of China (English)

    LI Huijia; LI Huiying

    2016-01-01

    Revealing the dynamics of community structure is of great concern for scientists from many fields.Specifically,how to quantify the dynamic details of soft community structure is a very interesting topic.In this paper,the authors propose a novel framework to study the scalable dynamic behavior of the soft community structure.First,the authors model the Potts dynamics to detect community structure using a "soft" Markov process.Then the soft stability of in a multiscale view is proposed to naturally uncover the local uniform behavior of spin values across multiple hierarchical levels.Finally,a new partition index is developed to detect fuzzy communities based on the stability and the dynamical information.Experiments on the both synthetically generated and real-world networks verify that the framework can be used to uncover hierarchical community structures effectively and efficiently.

  15. Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon

    Directory of Open Access Journals (Sweden)

    Schuster Martin

    2007-08-01

    Full Text Available Abstract Background Quorum-sensing regulation of gene expression in Pseudomonas aeruginosa is complex. Two interconnected acyl-homoserine lactone (acyl-HSL signal-receptor pairs, 3-oxo-dodecanoyl-HSL-LasR and butanoyl-HSL-RhlR, regulate more than 300 genes. The induction of most of the genes is delayed during growth of P. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. Many of these late genes can be induced by addition of signals early by using specific media conditions. While several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally. Results To better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors (in the presence of their cognate signals and another component that affects quorum sensing, the stationary phase sigma factor RpoS, early in growth. We determined the effect on target gene expression by microarray and real-time PCR analysis. Our results show that many target genes (e.g. lasB and hcnABC are directly responsive to receptor protein levels. Most genes (e.g. lasA, lecA, and phnAB, however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. The majority of promoters advanced by RhlR appeared to be regulated directly, which allowed us to build a RhlR consensus sequence. Conclusion The direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. The observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors

  16. Molecular characterization reveals the complexity of previously overlooked coral-exosymbiont interactions and the implications for coral-guild ecology

    Science.gov (United States)

    Rouzé, H.; Leray, M.; Magalon, H.; Penin, L.; Gélin, P.; Knowlton, N.; Fauvelot, C.

    2017-01-01

    Several obligate associate crabs and shrimps species may co-occur and interact within a single coral host, leading to patterns of associations that can provide essential ecological services. However, knowledge of the dynamics of interactions in this system is limited, partly because identifying species involved in the network remains challenging. In this study, we assessed the diversity of the decapods involved in exosymbiotic assemblages for juvenile and adult Pocillopora damicornis types α and β on reefs of New Caledonia and Reunion Island. This approach revealed complex patterns of association at regional and local scales with a prevalence of assemblages involving crab-shrimp partnerships. Furthermore, the distinction of two lineages in the snapping shrimp Alpheus lottini complex, rarely recognized in ecological studies, reveals a key role for cryptic diversity in structuring communities of mutualists. The existence of partnerships between species that occurred more commonly than expected by chance suggests an increased advantage for the host or a better adaptation of associated species to local environmental conditions. The consideration of cryptic diversity helps to accurately describe the complexity of interaction webs for diverse systems such as coral reefs, as well as the functional roles of dominant associated species for the persistence of coral populations. PMID:28358026

  17. Bioinformatic analysis of the neprilysin (M13 family of peptidases reveals complex evolutionary and functional relationships

    Directory of Open Access Journals (Sweden)

    Pinney John W

    2008-01-01

    Full Text Available Abstract Background The neprilysin (M13 family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2, which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. Results The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates

  18. Dynamic Modelling Reveals 'Hotspots' on the Pathway to Enzyme-Substrate Complex Formation.

    Directory of Open Access Journals (Sweden)

    Shane E Gordon

    2016-03-01

    Full Text Available Dihydrodipicolinate synthase (DHDPS catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions.

  19. Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins

    Directory of Open Access Journals (Sweden)

    Martijn A. Langereis

    2015-06-01

    Full Text Available Sialic acids (Sias, 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias. The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.

  20. Resistivity imaging reveals complex pattern of saltwater intrusion along Monterey coast

    Science.gov (United States)

    Goebel, Meredith; Pidlisecky, Adam; Knight, Rosemary

    2017-08-01

    Electrical Resistivity Tomography data were acquired along 40 km of the Monterey Bay coast in central California. These data resulted in electrical resistivity images to depths of approximately 280 m.b.s.l., which were used to understand the distribution of freshwater and saltwater in the subsurface, and factors controlling this distribution. The resulting resistivity sections were interpreted in conjunction with existing data sets, including well logs, seismic reflection data, geologic reports, hydrologic reports, and land use maps from the region. Interpretation of these data shows a complex pattern of saltwater intrusion resulting from geology, pumping, and recharge. The resistivity profiles were used to identify geological flow conduits and barriers such as palaeo-channels and faults, localized saltwater intrusion from individual pumping wells, infiltration zones of surface fresh and brackish water, and regions showing improvements in water quality due to management actions. The use of ERT data for characterizing the subsurface in this region has led to an understanding of the spatial distribution of freshwater and saltwater at a level of detail unattainable with the previously deployed traditional well based salinity mapping and monitoring techniques alone. Significant spatial variability in the extent and geometry of intrusion observed in the acquired data highlights the importance of adopting continuous subsurface characterization methods such as this one.

  1. Integrated intracellular metabolic profiling and pathway analysis approaches reveal complex metabolic regulation by Clostridium acetobutylicum.

    Science.gov (United States)

    Liu, Huanhuan; Huang, Di; Wen, Jianping

    2016-02-15

    Clostridium acetobutylicum is one of the most important butanol producing strains. However, environmental stress in the fermentation process usually leads to a lower yield, seriously hampering its industrialization. In order to systematically investigate the key intracellular metabolites that influence the strain growth and butanol production, and find out the critical regulation nodes, an integrated analysis approach has been carried out in this study. Based on the gas chromatography-mass spectrometry technology, the partial least square discriminant analysis and the pathway analysis, 40 metabolic pathways linked with 43 key metabolic nodes were identified. In-depth analysis showed that lots of amino acids metabolism promoted cell growth but exerted slight influence on butanol production, while sugar metabolism was favorable for cell growth but unfavorable for butanol synthesis. Besides, both lysine and succinic acid metabolism generated a complex effect on the whole metabolic network. Dicarboxylate metabolism exerted an indispensable role on cell growth and butanol production. Subsequently, rational feeding strategies were proposed to verify these conclusions and facilitate the butanol biosynthesis. Feeding amino acids, especially glycine and serine, could obviously improve cell growth while yeast extract, citric acid and ethylene glycol could significantly enhance both growth and butanol production. The feeding experiment confirmed that metabolic profiling combined with pathway analysis provided an accurate, reasonable and practical approach to explore the cellular metabolic activity and supplied a basis for improving butanol production. These strategies can also be extended for the production of other important bio-chemical compounds.

  2. Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

    Science.gov (United States)

    Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

    2016-10-20

    The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

  3. A Hidden State in Light-Harvesting Complex II Revealed By Multipulse Spectroscopy.

    Science.gov (United States)

    van Oort, Bart; van Grondelle, Rienk; van Stokkum, Ivo H M

    2015-04-23

    Light-harvesting complex II (LHCII) is pivotal both for collecting solar radiation for photosynthesis, and for protection against photodamage under high light intensities (via a process called nonphotochemical quenching, NPQ). Aggregation of LHCII is associated with fluorescence quenching, and is used as an in vitro model system of NPQ. However, there is no agreement on the nature of the quencher and on the validity of aggregation as a model system. Here, we use ultrafast multipulse spectroscopy to populate a quenched state in unquenched (unaggregated) LHCII. The state shows characteristic features of lutein and chlorophyll, suggesting that it is an excitonically coupled state between these two compounds. This state decays in approximately 10 ps, making it a strong competitor for photodamage and photochemical quenching. It is observed in trimeric and monomeric LHCII, upon re-excitation with pulses of different wavelengths and duration. We propose that this state is always present, but is scarcely populated under low light intensities. Under high light intensities it may become more accessible, e.g. by conformational changes, and then form a quenching channel. The same state may be the cause of fluorescence blinking observed in single-molecule spectroscopy of LHCII trimers, where a small subpopulation is in an energetically higher state where the pathway to the quencher opens up.

  4. Mitochondrial and nuclear DNA phylogenies reveal a complex evolutionary history in the Australasian robins (Passeriformes: Petroicidae).

    Science.gov (United States)

    Christidis, Les; Irestedt, Martin; Rowe, Dianne; Boles, Walter E; Norman, Janette A

    2011-12-01

    The Australasian robins (Petroicidae) comprise a relatively homogeneous group of small to medium-sized insectivorous birds. Their center of diversity is Australia and New Guinea (40 species) but seven species have managed to colonize geographically distant islands such as Tanimbar, New Britain, New Zealand, New Caledonia, Norfolk Island, Vanuatu, Solomon Islands, Fiji and Samoa. To resolve the evolutionary relationships within the Petroicidae, we here present the results of a phylogenetic analysis of sequence data from two mitochondrial genes (ND2, CO1) and one nuclear intron (β-Fibrinogen intron 5) for all 14 genera and 40 of the 46 currently recognized species. All phylogenetic analyses identified six primary lineages, treated here as subfamilies, within the Petroicidae: (1) Eopsaltriinae comprising Eopsaltria (excluding E. flaviventris), Tregellasia, Peneothello, Melanodryas, Poecilodryas and Heteromyias; (2) Drymodinae comprising Drymodes; (3) Microecinae comprising Microeca, Monachella and Eopsaltria flaviventris; (4) Petroicinae comprising Petroica and Eugerygone; (5) Pachycephalopsinae comprising Pachycephalopsis; and (6) Amalocichlinae comprising Amalocichla. The genera Eopsaltria, Microeca, Peneothello and Poecilodryas were found to be paraphyletic. Based on assessments of phylogenetic branching patterns and/or DNA divergence it also was apparent that Eopsaltriaaustralis, Tregellasialeucops, Melanodryascucullata, Heteromyiasalbispecularis, Drymodessupercilious and Microecaflavigaster may each comprise more than one species. The Petroicidae display a complex biogeographical history involving repeated radiations both within, and across Australia and New Guinea. It appears that dispersal into smaller islands such as New Britain, Tanimbar and the South Pacific has only been undertaken by species with a "flycatcher" body form.

  5. Hidden Markov models reveal complexity in the diving behaviour of short-finned pilot whales.

    Science.gov (United States)

    Quick, Nicola J; Isojunno, Saana; Sadykova, Dina; Bowers, Matthew; Nowacek, Douglas P; Read, Andrew J

    2017-03-31

    Diving behaviour of short-finned pilot whales is often described by two states; deep foraging and shallow, non-foraging dives. However, this simple classification system ignores much of the variation that occurs during subsurface periods. We used multi-state hidden Markov models (HMM) to characterize states of diving behaviour and the transitions between states in short-finned pilot whales. We used three parameters (number of buzzes, maximum dive depth and duration) measured in 259 dives by digital acoustic recording tags (DTAGs) deployed on 20 individual whales off Cape Hatteras, North Carolina, USA. The HMM identified a four-state model as the best descriptor of diving behaviour. The state-dependent distributions for the diving parameters showed variation between states, indicative of different diving behaviours. Transition probabilities were considerably higher for state persistence than state switching, indicating that dive types occurred in bouts. Our results indicate that subsurface behaviour in short-finned pilot whales is more complex than a simple dichotomy of deep and shallow diving states, and labelling all subsurface behaviour as deep dives or shallow dives discounts a significant amount of important variation. We discuss potential drivers of these patterns, including variation in foraging success, prey availability and selection, bathymetry, physiological constraints and socially mediated behaviour.

  6. Structures of GRP94-Nucleotide Complexes Reveal Mechanistic Differences Between the Hsp90 Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    Dollins, D.E.; Warren, J.J.; Immormino, R.M.; Gewirth, D.T.

    2009-06-02

    GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 {angstrom} crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a 'twisted V' conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.

  7. Hidden Markov models reveal complexity in the diving behaviour of short-finned pilot whales

    Science.gov (United States)

    Quick, Nicola J.; Isojunno, Saana; Sadykova, Dina; Bowers, Matthew; Nowacek, Douglas P.; Read, Andrew J.

    2017-01-01

    Diving behaviour of short-finned pilot whales is often described by two states; deep foraging and shallow, non-foraging dives. However, this simple classification system ignores much of the variation that occurs during subsurface periods. We used multi-state hidden Markov models (HMM) to characterize states of diving behaviour and the transitions between states in short-finned pilot whales. We used three parameters (number of buzzes, maximum dive depth and duration) measured in 259 dives by digital acoustic recording tags (DTAGs) deployed on 20 individual whales off Cape Hatteras, North Carolina, USA. The HMM identified a four-state model as the best descriptor of diving behaviour. The state-dependent distributions for the diving parameters showed variation between states, indicative of different diving behaviours. Transition probabilities were considerably higher for state persistence than state switching, indicating that dive types occurred in bouts. Our results indicate that subsurface behaviour in short-finned pilot whales is more complex than a simple dichotomy of deep and shallow diving states, and labelling all subsurface behaviour as deep dives or shallow dives discounts a significant amount of important variation. We discuss potential drivers of these patterns, including variation in foraging success, prey availability and selection, bathymetry, physiological constraints and socially mediated behaviour. PMID:28361954

  8. Statistical inference on genetic data reveals the complex demographic history of human populations in central Asia.

    Science.gov (United States)

    Palstra, Friso P; Heyer, Evelyne; Austerlitz, Frédéric

    2015-06-01

    The demographic history of modern humans constitutes a combination of expansions, colonizations, contractions, and remigrations. The advent of large scale genetic data combined with statistically refined methods facilitates inference of this complex history. Here we study the demographic history of two genetically admixed ethnic groups in Central Asia, an area characterized by high levels of genetic diversity and a history of recurrent immigration. Using Approximate Bayesian Computation, we infer that the timing of admixture markedly differs between the two groups. Admixture in the traditionally agricultural Tajiks could be dated back to the onset of the Neolithic transition in the region, whereas admixture in Kyrgyz is more recent, and may have involved the westward movement of Turkic peoples. These results are confirmed by a coalescent method that fits an isolation-with-migration model to the genetic data, with both Central Asian groups having received gene flow from the extremities of Eurasia. Interestingly, our analyses also uncover signatures of gene flow from Eastern to Western Eurasia during Paleolithic times. In conclusion, the high genetic diversity currently observed in these two Central Asian peoples most likely reflects the effects of recurrent immigration that likely started before historical times. Conversely, conquests during historical times may have had a relatively limited genetic impact. These results emphasize the need for a better understanding of the genetic consequences of transmission of culture and technological innovations, as well as those of invasions and conquests.

  9. Understanding the complexities of Salmonella-host crosstalk as revealed by in vivo model organisms.

    Science.gov (United States)

    Verma, Smriti; Srikanth, Chittur V

    2015-07-01

    Foodborne infections caused by non-typhoidal Salmonellae, such as Salmonella enterica serovar Typhimurium (ST), pose a major challenge in the developed and developing world. With constant rise of drug-resistant strains, understanding the epidemiology, microbiology, pathogenesis and host-pathogen interactions biology is a mandatory requirement to enable health systems to be ready to combat these illnesses. Patient data from hospitals, at least from some parts of the world, have aided in epidemiological understanding of ST-mediated disease. Most of the other aspects connected to Salmonella-host crosstalk have come from model systems that offer convenience, genetic tractability and low maintenance costs that make them extremely valuable tools. Complex model systems such as the bovine model have helped in understanding key virulence factors needed for infection. Simple systems such as fruit flies and Caenorhabditis elegans have aided in identification of novel virulence factors, host pathways and mechanistic details of interactions. Some of the path-breaking concepts of the field have come from mice model of ST colitis, which allows genetic manipulations as well as high degree of similarity to human counterpart. Together, they are invaluable for correlating in vitro findings of ST-induced disease progression in vivo. The current review is a compilation of various advances of ST-host interactions at cellular and molecular levels that has come from investigations involving model organisms. © 2015 International Union of Biochemistry and Molecular Biology.

  10. Functional Sphere Profiling Reveals the Complexity of Neuroblastoma Tumor-Initiating Cell Model

    Directory of Open Access Journals (Sweden)

    Aurélie Coulon

    2011-10-01

    Full Text Available Neuroblastoma (NB is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically “profile” the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

  11. Generalized additive models reveal the intrinsic complexity of wood formation dynamics.

    Science.gov (United States)

    Cuny, Henri E; Rathgeber, Cyrille B K; Kiessé, Tristan Senga; Hartmann, Felix P; Barbeito, Ignacio; Fournier, Meriem

    2013-04-01

    The intra-annual dynamics of wood formation, which involves the passage of newly produced cells through three successive differentiation phases (division, enlargement, and wall thickening) to reach the final functional mature state, has traditionally been described in conifers as three delayed bell-shaped curves followed by an S-shaped curve. Here the classical view represented by the 'Gompertz function (GF) approach' was challenged using two novel approaches based on parametric generalized linear models (GLMs) and 'data-driven' generalized additive models (GAMs). These three approaches (GFs, GLMs, and GAMs) were used to describe seasonal changes in cell numbers in each of the xylem differentiation phases and to calculate the timing of cell development in three conifer species [Picea abies (L.), Pinus sylvestris L., and Abies alba Mill.]. GAMs outperformed GFs and GLMs in describing intra-annual wood formation dynamics, showing two left-skewed bell-shaped curves for division and enlargement, and a right-skewed bimodal curve for thickening. Cell residence times progressively decreased through the season for enlargement, whilst increasing late but rapidly for thickening. These patterns match changes in cell anatomical features within a tree ring, which allows the separation of earlywood and latewood into two distinct cell populations. A novel statistical approach is presented which renews our understanding of xylogenesis, a dynamic biological process in which the rate of cell production interplays with cell residence times in each developmental phase to create complex seasonal patterns.

  12. Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol.

    Science.gov (United States)

    Santos, Nuno; Santos, Catarina; Valente, Teresa; Gortázar, Christian; Almeida, Virgílio; Correia-Neves, Margarida

    2015-01-01

    Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

  13. Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history.

    Science.gov (United States)

    Kandiba, Lina; Eichler, Jerry

    2013-08-01

    Sialic acids and the other nonulosonic acid sugars, legionaminic acid and pseudaminic acid, are nine carbon-containing sugars that can be detected as components of the glycans decorating proteins and other molecules in Eukarya and Bacteria. Yet, despite the prevalence of N-glycosylation in Archaea and the variety of sugars recruited for the archaeal version of this post-translational modification, only a single report of a nonulosonic acid sugar in an archaeal N-linked glycan has appeared. Hence, to obtain a clearer picture of nonulosonic acid sugar biosynthesis capability in Archaea, 122 sequenced genomes were scanned for the presence of genes involved in the biogenesis of these sugars. The results reveal that while Archaea and Bacteria share a common route of sialic acid biosynthesis, numerous archaeal nonulosonic acid sugar biosynthesis pathway components were acquired from elsewhere via various routes. Still, the limited number of Archaea encoding components involved in the synthesis of nonulosonic acid sugars implies that such saccharides are not major components of glycans in this domain.

  14. Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.

    Science.gov (United States)

    Gurley, Kyle A; Elliott, Sarah A; Simakov, Oleg; Schmidt, Heiko A; Holstein, Thomas W; Sánchez Alvarado, Alejandro

    2010-11-01

    Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid re-organization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of a new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time.

  15. Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus.

    Science.gov (United States)

    Derjuga, Anna; Gourley, Tania S; Holm, Teresa M; Heng, Henry H Q; Shivdasani, Ramesh A; Ahmed, Rafi; Andrews, Nancy C; Blank, Volker

    2004-04-01

    Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

  16. Single-molecule FRET reveals hidden complexity in a protein energy landscape.

    Science.gov (United States)

    Tsytlonok, Maksym; Ibrahim, Shehu M; Rowling, Pamela J E; Xu, Wenshu; Ruedas-Rama, Maria J; Orte, Angel; Klenerman, David; Itzhaki, Laura S

    2015-01-06

    Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

  17. Latent physiological factors of complex human diseases revealed by independent component analysis of clinarrays

    Directory of Open Access Journals (Sweden)

    Chen David P

    2010-10-01

    Full Text Available Abstract Background Diagnosis and treatment of patients in the clinical setting is often driven by known symptomatic factors that distinguish one particular condition from another. Treatment based on noticeable symptoms, however, is limited to the types of clinical biomarkers collected, and is prone to overlooking dysfunctions in physiological factors not easily evident to medical practitioners. We used a vector-based representation of patient clinical biomarkers, or clinarrays, to search for latent physiological factors that underlie human diseases directly from clinical laboratory data. Knowledge of these factors could be used to improve assessment of disease severity and help to refine strategies for diagnosis and monitoring disease progression. Results Applying Independent Component Analysis on clinarrays built from patient laboratory measurements revealed both known and novel concomitant physiological factors for asthma, types 1 and 2 diabetes, cystic fibrosis, and Duchenne muscular dystrophy. Serum sodium was found to be the most significant factor for both type 1 and type 2 diabetes, and was also significant in asthma. TSH3, a measure of thyroid function, and blood urea nitrogen, indicative of kidney function, were factors unique to type 1 diabetes respective to type 2 diabetes. Platelet count was significant across all the diseases analyzed. Conclusions The results demonstrate that large-scale analyses of clinical biomarkers using unsupervised methods can offer novel insights into the pathophysiological basis of human disease, and suggest novel clinical utility of established laboratory measurements.

  18. Ethiopian genetic diversity reveals linguistic stratification and complex influences on the Ethiopian gene pool.

    Science.gov (United States)

    Pagani, Luca; Kivisild, Toomas; Tarekegn, Ayele; Ekong, Rosemary; Plaster, Chris; Gallego Romero, Irene; Ayub, Qasim; Mehdi, S Qasim; Thomas, Mark G; Luiselli, Donata; Bekele, Endashaw; Bradman, Neil; Balding, David J; Tyler-Smith, Chris

    2012-07-13

    Humans and their ancestors have traversed the Ethiopian landscape for millions of years, and present-day Ethiopians show great cultural, linguistic, and historical diversity, which makes them essential for understanding African variability and human origins. We genotyped 235 individuals from ten Ethiopian and two neighboring (South Sudanese and Somali) populations on an Illumina Omni 1M chip. Genotypes were compared with published data from several African and non-African populations. Principal-component and STRUCTURE-like analyses confirmed substantial genetic diversity both within and between populations, and revealed a match between genetic data and linguistic affiliation. Using comparisons with African and non-African reference samples in 40-SNP genomic windows, we identified "African" and "non-African" haplotypic components for each Ethiopian individual. The non-African component, which includes the SLC24A5 allele associated with light skin pigmentation in Europeans, may represent gene flow into Africa, which we estimate to have occurred ~3 thousand years ago (kya). The non-African component was found to be more similar to populations inhabiting the Levant rather than the Arabian Peninsula, but the principal route for the expansion out of Africa ~60 kya remains unresolved. Linkage-disequilibrium decay with genomic distance was less rapid in both the whole genome and the African component than in southern African samples, suggesting a less ancient history for Ethiopian populations.

  19. Genome-Wide Analysis Revealed the Complex Regulatory Network of Brassinosteroid Effects in Photomorphogenesis

    Institute of Scientific and Technical Information of China (English)

    Li Song; Xiao-Yi Zhou; Li Li; Liang-Jiao Xue; Xi Yang; Hong-Wei Xue

    2009-01-01

    Light and brassinosteroids (BRs) have been proved to be crucial in regulating plant growth and development;however,the mechanism of how they synergistically function is still largely unknown.To explore the underlying mechanisms in photomorphogenesis,genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide (BL).Results showed that BR deficiency stimulates,while BL treatment suppresses,the expressions of lightresponsive genes and photomorphogenesis,confirming the negative effects of BR in photomorphogenesis.This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification,cellular metabolism and energy utilization during dark-light transition.Further analysis revealed that hormone biosynthesis and signaling-related genes,especially those of auxin,were altered under BL treatment or light stimuli,indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones.Additionally,suppressed ubiquitin-cycle pathway during light-dark transition hinted the presence of a complicated network among light,hormone,and protein degradation.The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway,which will help to elucidate the molecular mechanism of plant photomorphogenesis.

  20. The Glass Bead Game: experimental sintering of rhyolitic ash reveals complex behaviour of irregular multiphase particles

    Science.gov (United States)

    Pope, Robyn; Tuffen, Hugh; Owen, Jacqueline; James, Mike; Wadsworth, Fabian

    2016-04-01

    Sintering of magmatic particles profoundly influences the permeability, strength and compaction of fragmented magma in conduits and pyroclastic deposits. It involves initial rounding and agglutination of particles, with formation of inter-particle necks, followed by progressive viscous collapse of pores. The sintering behaviour of ash particles within tuffisite veins, which may mediate shallow outgassing in silicic eruptions, is of particular interest. Experimental studies on homogeneous synthetic glasses[1] have shown sintering rates to be time, temperature and grainsize-dependent, reflecting the influence of melt viscosity and pore-melt interfacial tension. A key objective is to reconstruct the temperature-time path of naturally sintered samples, so here we investigate the sintering of natural, angular ash fragments, to explore whether similar simple relationships emerge for more complex particle morphologies and internal textures. A glass-rich ballistic rhyolite bomb from the Cordón Caulle 2011-2012 eruption was ground and sieved to create various grainsizes of angular ash particles. The bomb contains 70 wt.% SiO2, 0.25 wt.% H2O, and ~30 vol.% crystal phases, as phenocrysts and microlites of plagioclase and pyroxenes. Particles were spread thinly over a sapphire surface in an N2-purged heated stage, and heated to 900, 1000 and 1100 °C, corresponding to melt viscosities of 105.4-107.7 Pa.s. Images were collected every 10-600 s during isothermal sintering over tens of minutes to hours. Quantitative image analysis using ImageJ allowed quantification of evolving particle size and shape (diameter and roundness) and inter-particle neck width. The rate of particle rounding was expected to be highest for smallest particles, and to decrease through time, but unlike synthetic glass bead experiments, no simple trends emerged. When the temporal evolution of particle roundness was tracked, some particles showed an unexpected, systematic increase in rounding rate with time

  1. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

    Science.gov (United States)

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-05-07

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

  2. Global terrestrial water storage connectivity revealed using complex climate network analyses

    Directory of Open Access Journals (Sweden)

    A. Y. Sun

    2015-04-01

    Full Text Available Terrestrial water storage (TWS exerts a key control in global water, energy, and biogeochemical cycles. Although certain causal relationships exist between precipitation and TWS, the latter also reflects impacts of anthropogenic activities. Thus, quantification of the spatial patterns of TWS will not only help to understand feedbacks between climate dynamics and hydrologic cycle, but also provide new model calibration constraints for improving the current land surface models. In this work, the connectivity of TWS is quantified using the climate network theory, which has received broad attention in the climate modeling community in recent years. Complex networks of TWS anomalies are built using two global TWS datasets, a remote-sensing product that is obtained from the Gravity Recovery and Climate Experiment (GRACE satellite mission, and a model-generated dataset from the global land data assimilation system's NOAH model (GLDAS-NOAH. Both datasets have 1 ° × 1 ° resolutions and cover most global land areas except for permafrost regions. TWS networks are built by first quantifying pairwise correlation among all valid TWS anomaly time series, and then applying a statistical cutoff threshold to retain only the most important features in the network. Basinwise network connectivity maps are used to illuminate connectivity of individual river basins with other regions. The constructed network degree centrality maps show TWS hotspots around the globe and the patterns are consistent with recent GRACE studies. Parallel analyses of networks constructed using the two datasets indicate that the GLDAS-NOAH model captures many of the spatial patterns shown by GRACE, although significant discrepancies exist in some regions. Thus, our results provide important insights for constraining land surface models, especially in data sparse regions.

  3. RNAseq Reveals Complex Response of Campylobacter jejuni to Ovine Bile and In vivo Gallbladder Environment.

    Science.gov (United States)

    Kreuder, Amanda J; Schleining, Jennifer A; Yaeger, Michael; Zhang, Qijing; Plummer, Paul J

    2017-01-01

    Colonization of the gallbladder by enteric pathogens such as Salmonella typhi, Listeria monocytogenes, and Campylobacter jejuni is thought to play a key role in transmission and persistence of these important zoonotic agents; however, little is known about the molecular mechanisms that allow for bacterial survival within this harsh environment. Recently, a highly virulent C. jejuni sheep abortion (SA) clone represented by the clinical isolate IA3902 has emerged as the dominant cause for sheep abortion in the United States. Previous studies have indicated that the C. jejuni clone SA can frequently be isolated from the gallbladders of otherwise healthy sheep, suggesting that the gallbladder may serve as an important reservoir for infection. To begin to understand the molecular mechanisms associated with survival in the host gallbladder, C. jejuni IA3902 was exposed for up to 24 h to both the natural ovine host in vivo gallbladder environment, as well as ovine bile in vitro. Following exposure, total RNA was isolated from the bile and high throughput deep sequencing of strand specific rRNA-depleted total RNA was used to characterize the transcriptome of IA3902 under these conditions. Our results demonstrated for the first time the complete transcriptome of C. jejuni IA3902 during exposure to an important host environment, the sheep gallbladder. Exposure to the host environment as compared to in vitro bile alone provided a more robust picture of the complexity of gene regulation required for survival in the host gallbladder. A subset of genes including a large number of protein coding genes as well as seven previously identified non-coding RNAs were confirmed to be differentially expressed within our data, suggesting that they may play a key role in adaptation upon exposure to these conditions. This research provides valuable insights into the molecular mechanisms that may be utilized by C. jejuni IA3902 to colonize and survive within the inhospitable gallbladder

  4. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing.

    Science.gov (United States)

    Griffen, Ann L; Beall, Clifford J; Campbell, James H; Firestone, Noah D; Kumar, Purnima S; Yang, Zamin K; Podar, Mircea; Leys, Eugene J

    2012-06-01

    Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1,393,579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.

  5. Rare Inherited and De Novo CNVs Reveal Complex Contributions to ASD Risk in Multiplex Families.

    Science.gov (United States)

    Leppa, Virpi M; Kravitz, Stephanie N; Martin, Christa Lese; Andrieux, Joris; Le Caignec, Cedric; Martin-Coignard, Dominique; DyBuncio, Christina; Sanders, Stephan J; Lowe, Jennifer K; Cantor, Rita M; Geschwind, Daniel H

    2016-09-01

    Rare mutations, including copy-number variants (CNVs), contribute significantly to autism spectrum disorder (ASD) risk. Although their importance has been established in families with only one affected child (simplex families), the contribution of both de novo and inherited CNVs to ASD in families with multiple affected individuals (multiplex families) is less well understood. We analyzed 1,532 families from the Autism Genetic Resource Exchange (AGRE) to assess the impact of de novo and rare CNVs on ASD risk in multiplex families. We observed a higher burden of large, rare CNVs, including inherited events, in individuals with ASD than in their unaffected siblings (odds ratio [OR] = 1.7), but the rate of de novo events was significantly lower than in simplex families. In previously characterized ASD risk loci, we identified 49 CNVs, comprising 24 inherited events, 19 de novo events, and 6 events of unknown inheritance, a significant enrichment in affected versus control individuals (OR = 3.3). In 21 of the 30 families (71%) in whom at least one affected sibling harbored an established ASD major risk CNV, including five families harboring inherited CNVs, the CNV was not shared by all affected siblings, indicating that other risk factors are contributing. We also identified a rare risk locus for ASD and language delay at chromosomal region 2q24 (implicating NR4A2) and another lower-penetrance locus involving inherited deletions and duplications of WWOX. The genetic architecture in multiplex families differs from that in simplex families and is complex, warranting more complete genetic characterization of larger multiplex ASD cohorts. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Nutrient export from catchments on forested landscapes reveals complex nonstationary and stationary climate signals

    Science.gov (United States)

    Mengistu, Samson G.; Quick, Christopher G.; Creed, Irena F.

    2013-06-01

    Headwater catchment hydrology and biogeochemistry are influenced by climate, including linear trends (nonstationary signals) and climate oscillations (stationary signals). We used an analytical framework to detect nonstationary and stationary signals in yearly time series of nutrient export [dissolved organic carbon (DOC), dissolved organic nitrogen (DON), nitrate (NO3--N), and total dissolved phosphorus (TDP)] in forested headwater catchments with differential water loading and water storage potential at the Turkey Lakes Watershed in Ontario, Canada. We tested the hypotheses that (1) climate has nonstationary and stationary effects on nutrient export, the combination of which explains most of the variation in nutrient export; (2) more metabolically active nutrients (e.g., DON, NO3--N, and TDP) are more sensitive to these signals; and (3) catchments with relatively low water loading and water storage capacity are more sensitive to these signals. Both nonstationary and stationary signals were identified, and the combination of both explained the majority of the variation in nutrient export data. More variation was explained in more labile nutrients (DON, NO3--N, and TDP), which were also more sensitive to climate signals. The catchment with low-water storage potential and low water loading was most sensitive to nonstationary and stationary climatic oscillations, suggesting that these hydrologic features are characteristic of the most effective sentinels of climate change. The observed complex links between climate change, climatic oscillations, and water nutrient fluxes in headwater catchments suggest that climate may have considerable influence on the productivity and biodiversity of surface waters, in addition to other drivers such as atmospheric pollution.

  7. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    Directory of Open Access Journals (Sweden)

    Holly J Atkinson

    2009-10-01

    Full Text Available The group of proteins that contain a thioredoxin (Trx fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  8. Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2015-07-01

    Full Text Available The RNA polymerase II (Pol II is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.

  9. Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

    Science.gov (United States)

    Zhang, Lu; Silva, Daniel-Adriano; Pardo-Avila, Fátima; Wang, Dong; Huang, Xuhui

    2015-07-01

    The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.

  10. Spatial patterns of African ungulate aggregation reveal complex but limited risk effects from reintroduced carnivores.

    Science.gov (United States)

    Moll, Remington J; Killion, Alexander K; Montgomery, Robert A; Tambling, Craig J; Hayward, Matt W

    2016-05-01

    The "landscape of fear" model, recently advanced in research on the non-lethal effects of carnivores on ungulates, predicts that prey will exhibit detectable antipredator behavior not only during risky times (i.e., predators in close proximity) but also in risky places (i.e., habitat where predators kill prey or tend to occur). Aggregation is an important antipredator response in numerous ungulate species, making it a useful metric to evaluate the strength and scope of the landscape of fear in a multi-carnivore, multi-ungulate system. We conducted ungulate surveys over a 2-year period in South Africa to test the influence of three broad-scale sources of variation in the landscape on spatial patterns in aggregation: (1) habitat structure, (2) where carnivores tended to occur (i.e., population-level utilization distributions), and (3) where carnivores tended to kill ungulate prey (i.e., probabilistic kill site maps). We analyzed spatial variation in aggregation for six ungulate species exposed to predation from recently reintroduced lion (Panthera leo) and spotted hyena (Crocuta crocuta). Although we did detect larger aggregations of ungulates in "risky places," these effects existed primarily for smaller-bodied (lion, an ambush (stalking) carnivore, had stronger influence on ungulate aggregation than the hyena, an active (coursing) carnivore. In addition, places where lions tended to kill prey had a greater effect on ungulate aggregation than places where lions tended to occur, but an opposing pattern existed for hyena. Our study reveals heterogeneity in the landscape of fear and suggests broad-scale risk effects following carnivore reintroduction only moderately influence ungulate aggregation size and vary considerably by predator hunting mode, type of predation risk, and prey species.

  11. DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan.

    Science.gov (United States)

    Ashfaq, Muhammad; Hebert, Paul D N; Mirza, M Sajjad; Khan, Arif M; Mansoor, Shahid; Shah, Ghulam S; Zafar, Yusuf

    2014-01-01

    Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Sequence diversity in the DNA barcode region (mtCOI-5') was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3' to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage "Pakistan". The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and "Pakistan" were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.

  12. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging.

    Science.gov (United States)

    Han, Meng; Giese, Guenter; Schmitz-Valckenberg, Steffen; Bindewald-Wittich, Almut; Holz, Frank G; Yu, Jiayi; Bille, Josef F; Niemz, Markolf H

    2007-01-01

    The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

  14. The Complex History of Alarcon Rise Mid-Ocean Ridge Rhyolite Revealed through Mineral Chemistry

    Science.gov (United States)

    Dreyer, B. M.; Portner, R. A.; Clague, D. A.; Daczko, N. R.; Castillo, P.; Bindeman, I. N.

    2014-12-01

    A suite of basalts to rhyolites recovered from the Alarcon Rise, the northern extension of the intermediate spreading-rate East Pacific Rise, provides an unparalleled test of established mechanisms for high-Si lava formation at ridges. Rhyolites are ≤35% phyric and poorly vesicular. Mafic xenoclasts are common, and plagioclase is the dominant phase. Olivine and clinopyroxene are also common, and orthopyroxene, FeTi-oxides, zircon, and rare pyrite blebs are present. Major and trace element glass data are consistent with MELTS models of fractional crystallization from a parental melt, but a diverse mineral population records added complexity. Olivine and plagioclase compositions are broadly consistent with models, with the exception of more variable Fo52-77 and An87-28 in a basaltic andesitic composition where pigeonite is predicted to replace olivine in the crystallizing assemblage between ~1085-1015°C; pigeonites analyzed in an andesite have lower Ca and Fe than predicted. Clinopyroxene variability generally increases with host melt SiO2, from Mg# 86-84 in basalts to Mg# 80-21 in rhyolites, and zoning is common with higher-MgO anhedral cores mantled by lower-MgO euhedral rims. Cooler magmas aided the preservation of disequilibrium and are supported by ~715-835°C Ti-in-zircon and ilmenite-magnetite thermometry in rhyolites. Despite a well-predicted liquid line of decent, multiple signals of chemical disequilibrium in intermediate to silicic melts support mixing of magmatic batches and/or assimilation of partially hydrous crust. Assimilation is permissible given δ18O values that are lower than expected solely from fractional crystallization (i.e., <6.3‰ at 77% SiO2), but assimilation extent is limited on the basis of δD ~82±8 and Pacific MORB-like 87Sr/86Sr. Zircon Hf-isotopes and trace element patterns support a juvenile oceanic crustal source. Whereas depleted Pacific MORB mantle source reservoir is supported by whole rock Sr-Nd isotopes, slight

  15. Temporal trends in mammal responses to fire reveals the complex effects of fire regime attributes.

    Science.gov (United States)

    Lindenmayer, David B; Blanchard, Wade; MacGregor, Christopher; Barton, Philip; Banks, Sam C; Crane, Mason; Michael, Damian; Okada, Sachiko; Berry, Laurence; Florance, Daniel; Gill, Malcolm

    2016-03-01

    guide management of when and where (prescribed) fire or, conversely, long-unburned vegetation is needed. The complexity of observed responses highlights the need for large reserves in which patterns of heterogeneity in fire regimes can be sustained in space and over time.

  16. Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain.

    Science.gov (United States)

    Xia, Yitian; Shang, Yuan; Zhang, Rongguang; Zhu, Jinwei

    2017-08-10

    The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  17. Complexity of genome evolution by segmental rearrangement in Brassica rapa revealed by sequence-level analysis

    Directory of Open Access Journals (Sweden)

    Paterson Andrew H

    2009-11-01

    Full Text Available Abstract Background The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level. Results We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events. Conclusion Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions

  18. Complex Crustal Structure Beneath Western Turkey Revealed by 3D Seismic Full Waveform Inversion (FWI)

    Science.gov (United States)

    Cubuk-Sabuncu, Yesim; Taymaz, Tuncay; Fichtner, Andreas

    2016-04-01

    We present a 3D radially anisotropic velocity model of the crust and uppermost mantle structure beneath the Sea of Marmara and surroundings based on the full waveform inversion method. The intense seismic activity and crustal deformation are observed in the Northwest Turkey due to transition tectonics between the strike-slip North Anatolian Fault (NAF) and the extensional Aegean region. We have selected and simulated complete waveforms of 62 earthquakes (Mw > 4.0) occurred during 2007-2015, and recorded at (Δ DAD). The spectral-element solver of the wave equation, SES3D algorithm, is used to simulate seismic wave propagation in 3D spherical coordinates (Fichtner, 2009). The Large Scale Seismic Inversion Framework (LASIF) workflow tool is also used to perform full seismic waveform inversion (Krischer et al., 2015). The initial 3D Earth model is implemented from the multi-scale seismic tomography study of Fichtner et al. (2013). Discrepancies between the observed and simulated synthetic waveforms are determined using the time-frequency misfits which allows a separation between phase and amplitude information (Fichtner et al., 2008). The conjugate gradient optimization method is used to iteratively update the initial Earth model when minimizing the misfit. The inversion is terminated after 19 iterations since no further advances are observed in updated models. Our analysis revealed shear wave velocity variations of the shallow and deeper crustal structure beneath western Turkey down to depths of ~35-40 km. Low shear wave velocity anomalies are observed in the upper and mid crustal depths beneath major fault zones located in the study region. Low velocity zones also tend to mark the outline of young volcanic areas. Our final 3D Earth model is tested using forward wave simulations of earthquakes (M ≥ 3.7) that were not used during the inversion process. The comparison of observed and synthetic seismograms, calculated by initial and final models, showed significant

  19. Isolation and sequencing of the cryIC-like delta endotoxin gene from ...

    African Journals Online (AJOL)

    use

    2012-02-16

    Feb 16, 2012 ... insecticidal toxin coding gene was cloned from an Egyptian isolate of B. thuringiensis. Sequence analyses of the cryIC ... insecticidal proteins into crop plants to protect them from ..... eggplant, cotton, cucumber and tomato, etc.

  20. Activity of Bacillus thuringiensis D(delta)-endotoxins against codling moth (Cydia pomonella L.) larvae

    NARCIS (Netherlands)

    Boncheva, R.; Dukiandjiev, S.; Minkov, I.; Maagd, de R.A.; Naimov, S.

    2006-01-01

    Solubilized protoxins of nine Cry1 and one hybrid Cry1 ¿-endotoxin from Bacillus thuringiensis were tested for their activity against larvae of the codling moth (Cydia pomonella L). Cry1Da was the most toxic, followed by Cry1Ab, Cry1Ba, and Cry1Ac, while Cry1Aa, Cry1Fa, Cry1Ia, and SN19 were still l

  1. Activity of wild-type and hybrid Bacillus thuringiensis delta-endotoxins against Agrotis ipsilon

    NARCIS (Netherlands)

    Maagd, de R.A.; Weemen-Hendriks, M.; Molthoff, J.W.; Naimov, S.

    2003-01-01

    Twelve Cry1 and two Cry9 ?-endotoxins fromBacillus thuringiensis were tested for their activity against black cutworm (Agrotis ipsilon).A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between

  2. Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates.

    Directory of Open Access Journals (Sweden)

    Bo Yuan

    2015-12-01

    Full Text Available Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100 is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases-about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual's susceptibility to acquiring disease-associated alleles.

  3. Fluorescence mapping of mitochondrial TIM23 complex reveals a water-facing, substrate-interacting helix surface.

    Science.gov (United States)

    Alder, Nathan N; Jensen, Robert E; Johnson, Arthur E

    2008-08-08

    Protein translocation across the mitochondrial inner membrane is mediated by the TIM23 complex. While its central component, Tim23, is believed to form a protein-conducting channel, the regions of this subunit that face the imported protein are unknown. To examine Tim23 structure and environment in intact membranes at high resolution, various derivatives, each with a single, environment-sensitive fluorescent probe positioned at a specific site, were assembled into functional TIM23 complexes in active mitochondria and analyzed by multiple spectral techniques. Probes placed sequentially throughout a transmembrane region that was identified by crosslinking as part of the protein-conducting channel revealed an alpha helix in an amphipathic environment. Probes on the aqueous-facing helical surface specifically underwent spectral changes during protein import, and their accessibility to hydrophilic quenching agents is considered in terms of channel gating. This approach has therefore provided an unprecedented view of a translocon channel structure in an intact, fully operational, membrane-embedded complex.

  4. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  5. Joint independent component analysis of structural and functional images reveals complex patterns of functional reorganisation in stroke aphasia.

    Science.gov (United States)

    Specht, Karsten; Zahn, Roland; Willmes, Klaus; Weis, Susanne; Holtel, Christiane; Krause, Bernd J; Herzog, Hans; Huber, Walter

    2009-10-01

    Previous functional activation studies in patients with aphasia have mostly relied on standard group comparisons of aphasic patients with healthy controls, which are biased towards regions showing the most consistent effects and disregard variability within groups. Groups of aphasic patients, however, show considerable variability with respect to lesion localisation and extent. Here, we use a novel method, joint independent component analysis (jICA), which allowed us to investigate abnormal patterns of functional activation with O(15)-PET during lexical decision in aphasic patients after middle cerebral artery stroke and to directly relate them to lesion information from structural MRI. Our results demonstrate that with jICA we could detect a network of compensatory increases in activity within bilateral anterior inferior temporal areas (BA20), which was not revealed by standard group comparisons. In addition, both types of analyses, jICA and group comparison, showed increased activity in the right posterior superior temporal gyrus in aphasic patients. Further, whereas standard analyses revealed no decreases in activation, jICA identified that left perisylvian lesions were associated with decreased activation of left posterior inferior frontal cortex, damaged in most patients, and extralesional remote decreases of activity within polar parts of the inferior temporal gyrus (BA38/20) and the occipital cortex (BA19). Taken together, our results demonstrate that jICA may be superior in revealing complex patterns of functional reorganisation in aphasia.

  6. Complex adaptive responses during antagonistic coevolution between Tribolium castaneum and its natural parasite Nosema whitei revealed by multiple fitness components

    Directory of Open Access Journals (Sweden)

    Bérénos Camillo

    2012-01-01

    Full Text Available Abstract Background Host-parasite coevolution can lead to local adaptation of either parasite or host if there is specificity (GxG interactions and asymmetric evolutionary potential between host and parasite. This has been demonstrated both experimentally and in field studies, but a substantial proportion of studies fail to detect such clear-cut patterns. One explanation for this is that adaptation can be masked by counter-adaptation by the antagonist. Additionally, genetic architecture underlying the interaction is often highly complex thus preventing specific adaptive responses. Here, we have employed a reciprocal cross-infection experiment to unravel the adaptive responses of two components of fitness affecting both parties with different complexities of the underlying genetic architecture (i.e. mortality and spore load. Furthermore, our experimental coevolution of hosts (Tribolium castaneum and parasites (Nosema whitei included paired replicates of naive hosts from identical genetic backgrounds to allow separation between host- and parasite-specific responses. Results In hosts, coevolution led to higher resistance and altered resistance profiles compared to paired control lines. Host genotype × parasite genotype interactions (GH × GP were observed for spore load (the trait of lower genetic complexity, but not for mortality. Overall parasite performance correlated with resistance of its matching host coevolution background reflecting a directional and unspecific response to strength of selection during coevolution. Despite high selective pressures exerted by the obligatory killing parasite, and host- and parasite-specific mortality profiles, no general pattern of local adaptation was observed, but one case of parasite maladaptation was consistently observed on both coevolved and control host populations. In addition, the use of replicate control host populations in the assay revealed one case of host maladaptation and one case of parasite

  7. Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

    Science.gov (United States)

    Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

    2014-04-01

    LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of

  8. Structure of DNA-Cationic Surfactant Complexes at Hydrophobically Modified and Hydrophilic Silica Surfaces as Revealed by Neutron Reflectometry

    DEFF Research Database (Denmark)

    Cardenas Gomez, Marite; Wacklin, Hanna; Campbell, Richard A.

    2011-01-01

    In this article, we discuss the structure and composition of mixed DNA-cationic surfactant adsorption layers on both hydrophobic and hydrophilic solid surfaces. We have focused on the effects of the bulk concentrations, the surfactant chain length, and the type solid surface on the interfacial...... layer structure (the location, coverage, and conformation the e DNA and surfactant molecules). Neutron reflectometry is the technique of choice for revealing the surface layer structure by means of selective deuteration. We start by studying the interfacial complexation of DNA...... with dodecyltrimethylammonium bromide (DTAB) and hexadecyltrimethylammonium bromide (CTAB) on hydrophobic surfaces, where we show that DNA molecules are located on top of a self-assembled surfactant monolayer, with the thickness of the DNA layer and the surfactant DNA ratio determined by the surface coverage of the underlying...

  9. Molecular basis for TANK recognition by TRAF1 revealed by the crystal structure of TRAF1/TANK complex.

    Science.gov (United States)

    Kim, Chang Min; Jeong, Jae-Hee; Son, Young-Jin; Choi, Jun-Hyuk; Kim, Sunghwan; Park, Hyun Ho

    2017-02-02

    Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a multifunctional adaptor protein involved in important processes of cellular signaling, including innate immunity and apoptosis. TRAF family member-associated NF-kappaB activator (TANK) has been identified as a competitive intracellular inhibitor of TRAF2 function. Although TRAF recognition by various receptors has been studied extensively in the field of TRAF-mediated biology, molecular and functional details of TANK recognition and interaction with TRAF1 have not been studied. In this study, we report the crystal structure of the TRAF1/TANK peptide complex. Quantitative interaction experiments showed that TANK peptide interacts with both TRAF1 and TRAF2 with similar affinity in a micromolar range. Our structural study also reveals that TANK binds TRAF1 using a minor minimal consensus motif for TRAF binding, Px(Q/E)xT.

  10. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    Science.gov (United States)

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

    2016-10-04

    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

  11. Reconstitution of the Escherichia coli cell division ZipA-FtsZ complexes in nanodiscs as revealed by electron microscopy.

    Science.gov (United States)

    Hernández-Rocamora, Víctor M; García-Montañés, Concepción; Rivas, Germán; Llorca, Oscar

    2012-12-01

    ZipA is an element of the bacterial division ring complex that provides an anchor to the membrane to FtsZ, a GTPase ancestor of tubulin. In vitro reconstitution and characterization of these interactions is challenged by the difficulty to integrate a physiological membrane environment. Here a single copy of the full-length ZipA protein from Escherichia coli incorporated into phospholipid bilayer nanodiscs (Nd-ZipA) has been visualized using negative-staining electron microscopy (EM). The EM images reveal the presence of discs, mostly organized in two distinct populations of 11 and 13nm in diameter. The globular FtsZ-binding C-terminal domain of ZipA (ZBD) was not visible in 3D reconstructions of Nd-ZipA or 2D averages, suggesting that this domain is separated from the membrane by the large flexible domain connecting the N-terminal trans-membrane region to the ZBD. We tested if Nd-ZipA were appropriate models for the in vitro reconstitution of ZipA-FtsZ interactions. First we observed that the ZBD region of ZipA was accessible for the interaction with other proteins in the context of the nanodisc, as revealed by its recognition by specific antibodies. In addition, Nd-ZipA attached to carbon coated EM grids, but not empty nanodiscs, were able to capture FtsZ filaments without inducing significant filament bundling, consistent with a model in which FtsZ filaments are loosely attached to the cell-membrane. These observations are compatible with the plastic nature of the ZipA-FtsZ complexes formed at the membrane, evidenced in the moderate binding affinity of Nd-ZipA to FtsZ oligomers and polymers recently measured.

  12. The Paleoproterozoic Singo granite in south-central Uganda revealed as a nested igneous ring complex using geophysical data

    Science.gov (United States)

    Abdelsalam, Mohamed G.; Katumwehe, Andrew B.; Atekwana, Estella A.; Le Pera, Alan K.; Achang, Mercy

    2016-04-01

    We used high-resolution airborne magnetic and radiometric data and satellite gravity data to investigate the form of occurrence of the Paleoproterozoic Singo granite in west-central Uganda. This granitic body covers an area of ∼700 km2, intrudes Paleoproterozoic crystalline rocks and overlain by Paleoproterozoic-Mesoproterozoic sedimentary rocks, both of which belong to the Rwenzori terrane, and it is host to hydrothermally-formed economic minerals such as gold and tungsten. Our analysis provided unprecedented geometrical details of the granitic body and revealed the following: (1) the margins of the Singo granite are characterized by a higher magnetic signature compared to the interior of the granitic body as well as the surroundings. These anomalies are apparent in both the total magnetic field and horizontal derivative images and define eight overlapping ring features. (2) the depth continuation of these magnetic anomalies define outward but steeply-dipping features as indicated by the tilt images extracted from the airborne magnetic data. This is further supported by forward modeling of the magnetic and gravity data. (3) the Singo granite is characterized by relatively high and evenly-distributed equivalent concentration of Uranium (eU) and Thorium (eTh) compared to the surroundings and this is apparent in the Potassium (K)-eTh-eU radiometric ternary image. (4) the granitic body is defined by a gravity low anomaly that persisted to a depth of three km as shown by the Bouguer anomaly image and its five km upward continuation. We used these observations to identify this granitic body as a nested igneous ring complex and we refer to it as the Singo Igneous Ring Complex (SIRC). We further interpreted the eight ring structures as individual igneous ring complexes aligned in an E-W and NE-SW direction and these were developed due to repeated calderas collapse. Additionally, we interpreted the ring-shaped magnetic anomalies as due to hydrothermally-altered margins

  13. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    Science.gov (United States)

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  14. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    Science.gov (United States)

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways. PMID:25275011

  15. Crystal Structure of the FGFR4/LY2874455 Complex Reveals Insights into the Pan-FGFR Selectivity of LY2874455.

    Science.gov (United States)

    Wu, Daichao; Guo, Ming; Philips, Michael A; Qu, Lingzhi; Jiang, Longying; Li, Jun; Chen, Xiaojuan; Chen, Zhuchu; Chen, Lin; Chen, Yongheng

    2016-01-01

    Aberrant FGFR4 signaling has been documented abundantly in various human cancers. The majority of FGFR inhibitors display significantly reduced potency toward FGFR4 compared to FGFR1-3. However, LY2874455 has similar inhibition potency for FGFR1-4 with IC50 less than 6.4 nM. To date, there is no published crystal structure of LY2874455 in complex with any kinase. To better understand the pan-FGFR selectivity of LY2874455, we have determined the crystal structure of the FGFR4 kinase domain bound to LY2874455 at a resolution of 2.35 Å. LY2874455, a type I inhibitor for FGFR4, binds to the ATP-binding pocket of FGFR4 in a DFG-in active conformation with three hydrogen bonds and a number of van der Waals contacts. After alignment of the kinase domain sequence of 4 FGFRs, and superposition of the ATP binding pocket of 4 FGFRs, our structural analyses reveal that the interactions of LY2874455 to FGFR4 are largely conserved in 4 FGFRs, explaining at least partly, the broad inhibitory activity of LY2874455 toward 4 FGFRs. Consequently, our studies reveal new insights into the pan-FGFR selectivity of LY2874455 and provide a structural basis for developing novel FGFR inhibitors that target FGFR1-4 broadly.

  16. Complex within a Complex: Integrative Taxonomy Reveals Hidden Diversity in Cicadetta brevipennis (Hemiptera: Cicadidae) and Unexpected Relationships with a Song Divergent Relative

    Science.gov (United States)

    Hertach, Thomas; Puissant, Stéphane; Gogala, Matija; Trilar, Tomi; Hagmann, Reto; Baur, Hannes; Kunz, Gernot; Wade, Elizabeth J.; Loader, Simon P.; Simon, Chris; Nagel, Peter

    2016-01-01

    Multiple sources of data in combination are essential for species delimitation and classification of difficult taxonomic groups. Here we investigate a cicada taxon with unusual cryptic diversity and we attempt to resolve seemingly contradictory data sets. Cicada songs act as species-specific premating barriers and have been used extensively to reveal hidden taxonomic diversity in morphologically similar species. The Palaearctic Cicadetta montana species complex is an excellent example where distinct song patterns have disclosed multiple recently described species. Indeed, two taxa turned out to be especially diverse in that they form a “complex within the complex”: the Cicadetta cerdaniensis song group (four species studied previously) and Cicadetta brevipennis (examined in details here). Based on acoustic, morphological, molecular, ecological and spatial data sampled throughout their broad European distribution, we find that Cicadetta brevipennis s. l. comprises five lineages. The most distinct lineage is identified as Cicadetta petryi Schumacher, 1924, which we re-assign to the species level. Cicadetta brevipennis litoralis Puissant & Hertach ssp. n. and Cicadetta brevipennis hippolaidica Hertach ssp. n. are new to science. The latter hybridizes with Cicadetta brevipennis brevipennis Fieber, 1876 at a zone inferred from intermediate song patterns. The fifth lineage requires additional investigation. The C. cerdaniensis and the C. brevipennis song groups exhibit characteristic, clearly distinct basic song patterns that act as reproductive barriers. However, they remain completely intermixed in the Bayesian and maximum likelihood COI and COII mitochondrial DNA phylogenies. The closest relative of each of the four cerdaniensis group species is a brevipennis group taxon. In our favoured scenario the phylogenetic pairs originated in common Pleistocene glacial refuges where the taxa speciated and experienced sporadic inter-group hybridization leading to extensive

  17. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.

    Directory of Open Access Journals (Sweden)

    Peter Rellos

    Full Text Available UNLABELLED: Long-term potentiation (LTP, a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM-dependent kinase II (CaMKII. CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

  18. Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism.

    Science.gov (United States)

    Gregg, Katie J; Suits, Michael D L; Deng, Lehua; Vocadlo, David J; Boraston, Alisdair B

    2015-10-16

    O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.

  19. RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions.

    Directory of Open Access Journals (Sweden)

    Olga Østrup

    Full Text Available Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA. While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv and in vitro produced (ivt porcine embryos before (2-cell stage and after (late 4-cell stage EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery, protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism, different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and

  20. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.

    Directory of Open Access Journals (Sweden)

    Benjamin G Kopek

    2007-09-01

    Full Text Available Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules, which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

  1. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  2. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis.

    Directory of Open Access Journals (Sweden)

    Guoying Dong

    Full Text Available H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G. Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.

  3. Ancient DNA reveals prehistoric gene-flow from siberia in the complex human population history of North East Europe.

    Science.gov (United States)

    Der Sarkissian, Clio; Balanovsky, Oleg; Brandt, Guido; Khartanovich, Valery; Buzhilova, Alexandra; Koshel, Sergey; Zaporozhchenko, Valery; Gronenborn, Detlef; Moiseyev, Vyacheslav; Kolpakov, Eugen; Shumkin, Vladimir; Alt, Kurt W; Balanovska, Elena; Cooper, Alan; Haak, Wolfgang

    2013-01-01

    North East Europe harbors a high diversity of cultures and languages, suggesting a complex genetic history. Archaeological, anthropological, and genetic research has revealed a series of influences from Western and Eastern Eurasia in the past. While genetic data from modern-day populations is commonly used to make inferences about their origins and past migrations, ancient DNA provides a powerful test of such hypotheses by giving a snapshot of the past genetic diversity. In order to better understand the dynamics that have shaped the gene pool of North East Europeans, we generated and analyzed 34 mitochondrial genotypes from the skeletal remains of three archaeological sites in northwest Russia. These sites were dated to the Mesolithic and the Early Metal Age (7,500 and 3,500 uncalibrated years Before Present). We applied a suite of population genetic analyses (principal component analysis, genetic distance mapping, haplotype sharing analyses) and compared past demographic models through coalescent simulations using Bayesian Serial SimCoal and Approximate Bayesian Computation. Comparisons of genetic data from ancient and modern-day populations revealed significant changes in the mitochondrial makeup of North East Europeans through time. Mesolithic foragers showed high frequencies and diversity of haplogroups U (U2e, U4, U5a), a pattern observed previously in European hunter-gatherers from Iberia to Scandinavia. In contrast, the presence of mitochondrial DNA haplogroups C, D, and Z in Early Metal Age individuals suggested discontinuity with Mesolithic hunter-gatherers and genetic influx from central/eastern Siberia. We identified remarkable genetic dissimilarities between prehistoric and modern-day North East Europeans/Saami, which suggests an important role of post-Mesolithic migrations from Western Europe and subsequent population replacement/extinctions. This work demonstrates how ancient DNA can improve our understanding of human population movements across

  4. Ancient DNA reveals prehistoric gene-flow from siberia in the complex human population history of North East Europe.

    Directory of Open Access Journals (Sweden)

    Clio Der Sarkissian

    Full Text Available North East Europe harbors a high diversity of cultures and languages, suggesting a complex genetic history. Archaeological, anthropological, and genetic research has revealed a series of influences from Western and Eastern Eurasia in the past. While genetic data from modern-day populations is commonly used to make inferences about their origins and past migrations, ancient DNA provides a powerful test of such hypotheses by giving a snapshot of the past genetic diversity. In order to better understand the dynamics that have shaped the gene pool of North East Europeans, we generated and analyzed 34 mitochondrial genotypes from the skeletal remains of three archaeological sites in northwest Russia. These sites were dated to the Mesolithic and the Early Metal Age (7,500 and 3,500 uncalibrated years Before Present. We applied a suite of population genetic analyses (principal component analysis, genetic distance mapping, haplotype sharing analyses and compared past demographic models through coalescent simulations using Bayesian Serial SimCoal and Approximate Bayesian Computation. Comparisons of genetic data from ancient and modern-day populations revealed significant changes in the mitochondrial makeup of North East Europeans through time. Mesolithic foragers showed high frequencies and diversity of haplogroups U (U2e, U4, U5a, a pattern observed previously in European hunter-gatherers from Iberia to Scandinavia. In contrast, the presence of mitochondrial DNA haplogroups C, D, and Z in Early Metal Age individuals suggested discontinuity with Mesolithic hunter-gatherers and genetic influx from central/eastern Siberia. We identified remarkable genetic dissimilarities between prehistoric and modern-day North East Europeans/Saami, which suggests an important role of post-Mesolithic migrations from Western Europe and subsequent population replacement/extinctions. This work demonstrates how ancient DNA can improve our understanding of human population

  5. Molecular epidemiology reveals genetic diversity amongst isolates of the Cryptococcus neoformans/C. gattii species complex in Thailand.

    Directory of Open Access Journals (Sweden)

    Sirada Kaocharoen

    Full Text Available To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9% were isolated from males (mean age of 37.97 years, 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates. In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates, VNIV (0.2%; 100% environmental isolate, VGI (0.2%; 100% clinical isolate and VGII (2.4%; 100% clinical isolates were found less frequently. Multilocus Sequence Type (MLST analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.

  6. Transcriptome, carbohydrate and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

    Directory of Open Access Journals (Sweden)

    Martin Andreas Bauerfeind

    2015-07-01

    Full Text Available Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12°C vs. 16°C affect growth of the sensitive Petunia hybrida cultivar `SweetSunshine Williams´, the transcriptome, carbohydrate metabolism and phytohormone homeostasis were monitored in aerial plant parts over four weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After three weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently

  7. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria.

    Science.gov (United States)

    Hoppins, Suzanne; Collins, Sean R; Cassidy-Stone, Ann; Hummel, Eric; Devay, Rachel M; Lackner, Laura L; Westermann, Benedikt; Schuldiner, Maya; Weissman, Jonathan S; Nunnari, Jodi

    2011-10-17

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

  8. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

    Directory of Open Access Journals (Sweden)

    Jie Qiu

    Full Text Available Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou and a wild line (Lanxi 1 collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1 no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2 besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3 high heterozygous rates (0.19-0.49 were observed in several semi-wild lines; and (4 over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

  9. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

    Science.gov (United States)

    Qiu, Jie; Wang, Yu; Wu, Sanling; Wang, Ying-Ying; Ye, Chu-Yu; Bai, Xuefei; Li, Zefeng; Yan, Chenghai; Wang, Weidi; Wang, Ziqiang; Shu, Qingyao; Xie, Jiahua; Lee, Suk-Ha; Fan, Longjiang

    2014-01-01

    Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou) and a wild line (Lanxi 1) collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1) no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2) besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3) high heterozygous rates (0.19-0.49) were observed in several semi-wild lines; and (4) over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

  10. A comprehensive custom panel design for routine hereditary cancer testing: preserving control, improving diagnostics and revealing a complex variation landscape.

    Science.gov (United States)

    Castellanos, Elisabeth; Gel, Bernat; Rosas, Inma; Tornero, Eva; Santín, Sheila; Pluvinet, Raquel; Velasco, Juan; Sumoy, Lauro; Del Valle, Jesús; Perucho, Manuel; Blanco, Ignacio; Navarro, Matilde; Brunet, Joan; Pineda, Marta; Feliubadaló, Lidia; Capellá, Gabi; Lázaro, Conxi; Serra, Eduard

    2017-01-04

    We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.

  11. Monophyly, distance and character-based multigene barcoding reveal extraordinary cryptic diversity in Nassarius: a complex and dangerous community.

    Directory of Open Access Journals (Sweden)

    Shanmei Zou

    Full Text Available BACKGROUND: Correct identification and cryptic biodiversity revelation for marine organisms are pressing since the marine life is important in maintaining the balance of ecological system and is facing the problem of biodiversity crisis or food safety. DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Nassarius, the common mudsnail, plays an important role in marine environment and has problem in food safety, but the classification of it is quite confused because of the complex morphological diversity. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a comprehensive barcoding analysis of 22 Nassarius species. We integrated the mitochondrial and nuclear sequences and the morphological characters to determine 13 Nassarius species studied and reveal four cryptic species and one pair synonyms. Distance, monophyly, and character-based barcoding methods were employed. CONCLUSIONS/SIGNIFICANCE: Such successful identification and unexpected cryptic discovery is significant for Nassarius in food safety and species conversation and remind us to pay more attention to the hidden cryptic biodiversity ignored in marine life. Distance, monophyly, and character-based barcoding methods are all very helpful in identification but the character-based method shows some advantages.

  12. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W [SVIMR-A; (Hanson); (Heidelberg); (Melbourne)

    2012-04-17

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  13. Complex organizational structure of the genome revealed by genome-wide analysis of single and alternative promoters in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Zhu Qianqian

    2009-01-01

    Full Text Available Abstract Background The promoter is a critical necessary transcriptional cis-regulatory element. In addition to its role as an assembly site for the basal transcriptional apparatus, the promoter plays a key part in mediating temporal and spatial aspects of gene expression through differential binding of transcription factors and selective interaction with distal enhancers. Although many genes have multiple promoters, little attention has been focused on how these relate to one another; nor has much study been directed at relationships between promoters of adjacent genes. Results We have undertaken a systematic investigation of Drosophila promoters. We divided promoters into three groups: unique promoters, first alternative promoters (the most 5' of a gene's multiple promoters, and downstream alternative promoters (the remaining alternative promoters 3' to the first. We observed distinct nucleotide distribution and sequence motif preferences among these three classes. We also investigated the promoters of neighboring genes and found that a greater than expected number of adjacent genes have similar sequence motif profiles, which may allow the genes to be regulated in a coordinated fashion. Consistent with this, there is a positive correlation between similar promoter motifs and related gene expression profiles for these genes. Conclusions Our results suggest that different regulatory mechanisms may apply to each of the three promoter classes, and provide a mechanism for "gene expression neighborhoods," local clusters of co-expressed genes. As a whole, our data reveal an unexpected complexity of genomic organization at the promoter level with respect to both alternative and neighboring promoters.

  14. Tetrapositive plutonium, neptunium, uranium, and thorium coordination complexes: chemistry revealed by electron transfer and collision induced dissociation.

    Science.gov (United States)

    Gong, Yu; Tian, Guoxin; Rao, Linfeng; Gibson, John K

    2014-04-17

    The Pu(4+), Np(4+), and U(4+) ions, which have large electron affinities of ∼34.6, ∼33.6, and ∼32.6 eV, respectively, were stabilized from solution to the gas phase upon coordination by three neutral tetramethyl-3-oxa-glutaramide ligands (TMOGA). Both collision induced dissociation (CID) and electron transfer dissociation (ETD) of Pu(TMOGA)3(4+) reveal the propensity for reduction of Pu(IV) to Pu(III), by loss of TMOGA(+) in CID and by simple electron transfer in ETD. The reduction of Pu(IV) is in distinct contrast to retention of Th(IV) in both CID and ETD of Th(TMOGA)3(4+), where only the C-Oether bond cleavage product was observed. U(TMOGA)3(4+) behaves similarly to Th(TMOGA)3(4+) upon CID and ETD, while the fragmentation patterns of Np(TMOGA)3(4+) lie between those of Pu(TMOGA)3(4+) and U(TMOGA)3(4+). It is notable that the gas-phase fragmentation behaviors of these exceptional tetrapositive complexes parallel fundamental differences in condensed phase chemistry within the actinide series, specifically the tendency for reduction from the IV to III oxidation states.

  15. Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome.

    Science.gov (United States)

    Sun, Ji; MacKinnon, Roderick

    2017-06-01

    KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. An integrated RNA-Seq and network study reveals a complex regulation process of rice embryo during seed germination.

    Science.gov (United States)

    Wei, Ting; He, Zilong; Tan, XinYu; Liu, Xue; Yuan, Xiao; Luo, Yingfeng; Hu, Songnian

    2015-08-14

    Seed germination is a crucial stage for plant development and agricultural production. To investigate its complex regulation process, the RNA-Seq study of rice embryo was conducted at three time points of 0, 12 and 48 h post imbibition (HPI). Dynamic transcriptional alterations were observed, especially in the early stage (0-12 HPI). Seed related genes, especially those encoding desiccation inducible proteins and storage reserves in embryo, decreased drastically after imbibition. The expression profiles of phytohormone related genes indicated distinct roles of abscisic acid (ABA), gibberellin (GA) and brassinosteroid (BR) in germination. Moreover, network analysis revealed the importance of protein phosphorylation in phytohormone interactions. Network and gene ontology (GO) analyses suggested that transcription factors (TFs) played a regulatory role in functional transitions during germination, and the enriched TF families at 0 HPI implied a regulation of epigenetic modification in dry seeds. In addition, 35 germination-specific TF genes in embryo were identified and seven genes were verified by qRT-PCR. Besides, enriched TF binding sites (TFBSs) supported physiological changes in germination. Overall, this study expands our comprehensive knowledge of multiple regulation factors underlying rice seed germination.

  17. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    Energy Technology Data Exchange (ETDEWEB)

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S., E-mail: ngk@ucalgary.ca [University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4 (Canada)

    2013-05-01

    The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  18. Whole genome sequencing reveals complex evolution patterns of multidrug-resistant Mycobacterium tuberculosis Beijing strains in patients.

    Directory of Open Access Journals (Sweden)

    Matthias Merker

    Full Text Available Multidrug-resistant (MDR Mycobacterium tuberculosis complex (MTBC strains represent a major threat for tuberculosis (TB control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A and nine (Patient B polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and

  19. Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex

    Science.gov (United States)

    Wu, Yi-Min; Wang, Chun-Hsiung; Chang, Jen-wei; Chen, Yi-yun; Miyazaki, Naoyuki; Murata, Kazuyoshi; Nagayama, Kuniaki; Chang, Wei-Hau

    2013-12-01

    Cryo-electron microscopy (cryo-EM) has become a powerful technique for obtaining near atomic structures for large protein assemblies or large virus particles, but the application to protein particles smaller than 200-300 kDa has been hampered by the feeble phase contrast obtained for such small samples and the limited number of electrons tolerated by them without incurring excessive radiation damage. By implementing a thin-film quarter-wave phase plate to a cryo-EM, Nagayama, one of the present authors, has recently restored the long-lost very low spatial frequencies, generating in-focus phase contrast superior to that of conventional defocusing phase contrast, and successfully applied the so-called Zernike phase-plate cryo-EM to target various biological samples in native state. Nevertheless, the sought-after goal of using enhanced phase contrast to reveal a native protein as small as 100 kDa waits to be realized. Here, we report a study in which 200 kV Zernike phase-plate cryo-EM with a plate cut-on periodicity of 36 nm was applied to visualize 100 kDa components of various protein complexes, including the small domains on the surface of an icosahedral particle of ˜38 nm derived from the dragon grouper nervous necrosis virus (DGNNV) and the labile sub-complex dissociated from yeast RNA polymerase III of 17 nm. In the former case, we observed a phase contrast reversal phenomenon at the centre of the icosahedral particle and traced its root cause to the near matching of the cut-on size and the particle size. In summary, our work has demonstrated that Zernike phase-plate implementation can indeed expand the size range of proteins that can be successfully investigated by cryo-EM, opening the door for countless proteins. Finally, we briefly discuss the possibility of using a transfer lens system to enlarge the cut-on periodicity without further miniaturizing the plate pinhole.

  20. Multilocus phylogeny of the avian family Alaudidae (larks) reveals complex morphological evolution, non-monophyletic genera and hidden species diversity.

    Science.gov (United States)

    Alström, Per; Barnes, Keith N; Olsson, Urban; Barker, F Keith; Bloomer, Paulette; Khan, Aleem Ahmed; Qureshi, Masood Ahmed; Guillaumet, Alban; Crochet, Pierre-André; Ryan, Peter G

    2013-12-01

    The Alaudidae (larks) is a large family of songbirds in the superfamily Sylvioidea. Larks are cosmopolitan, although species-level diversity is by far largest in Africa, followed by Eurasia, whereas Australasia and the New World have only one species each. The present study is the first comprehensive phylogeny of the Alaudidae. It includes 83.5% of all species and representatives from all recognised genera, and was based on two mitochondrial and three nuclear loci (in total 6.4 kbp, although not all loci were available for all species). In addition, a larger sample, comprising several subspecies of some polytypic species was analysed for one of the mitochondrial loci. There was generally good agreement in trees inferred from different loci, although some strongly supported incongruences were noted. The tree based on the concatenated multilocus data was overall well resolved and well supported by the data. We stress the importance of performing single gene as well as combined data analyses, as the latter may obscure significant incongruence behind strong nodal support values. The multilocus tree revealed many unpredicted relationships, including some non-monophyletic genera (Calandrella, Mirafra, Melanocorypha, Spizocorys). The tree based on the extended mitochondrial data set revealed several unexpected deep divergences between taxa presently treated as conspecific (e.g. within Ammomanes cinctura, Ammomanes deserti, Calandrella brachydactyla, Eremophila alpestris), as well as some shallow splits between currently recognised species (e.g. Certhilauda brevirostris-C. semitorquata-C. curvirostris; Calendulauda barlowi-C. erythrochlamys; Mirafra cantillans-M. javanica). Based on our results, we propose a revised generic classification, and comment on some species limits. We also comment on the extraordinary morphological adaptability in larks, which has resulted in numerous examples of parallel evolution (e.g. in Melanocorypha mongolica and Alauda leucoptera [both

  1. Ecomorph or Endangered Coral? DNA and Microstructure Reveal Hawaiian Species Complexes: Montipora dilatata/flabellata/turgescens & M. patula/verrilli

    Science.gov (United States)

    Forsman, Zac H.; Concepcion, Gregory T.; Haverkort, Roxanne D.; Shaw, Ross W.; Maragos, James E.; Toonen, Robert J.

    2010-01-01

    M. dilatata, M. flabellata, and M. patula and 80 other scleractinian corals were petitioned to be listed under the US Endangered Species Act (ESA), which would have major conservation implications. One of the difficulties with this evaluation is that reproductive boundaries between morphologically defined coral species are often permeable, and morphology can be wildly variable. We examined genetic and morphological variation in Hawaiian Montipora with a suite of molecular markers (mitochondrial: COI, CR, Cyt-B, 16S, ATP6; nuclear: ATPsβ, ITS) and microscopic skeletal measurements. Mitochondrial markers and the ITS region revealed four distinct clades: I) M. patula/M. verrilli, II) M. cf. incrassata, III) M. capitata, IV) M. dilatata/M. flabellata/M. cf. turgescens. These clades are likely to occur outside of Hawai'i according to mitochondrial control region haplotypes from previous studies. The ATPsβ intron data showed a pattern often interpreted as resulting from hybridization and introgression; however, incomplete lineage sorting may be more likely since the multicopy nuclear ITS region was consistent with the mitochondrial data. Furthermore, principal components analysis (PCA) of skeletal microstructure was concordant with the mitochondrial clades, while nominal taxa overlapped. The size and shape of verrucae or papillae contributed most to identifying groups, while colony-level morphology was highly variable. It is not yet clear if these species complexes represent population-level variation or incipient speciation (CA<1MYA), two alternatives that have very different conservation implications. This study highlights the difficulty in understanding the scale of genetic and morphological variation that corresponds to species as opposed to population-level variation, information that is essential for conservation and for understanding coral biodiversity. PMID:21151995

  2. Ecomorph or endangered coral? DNA and microstructure reveal hawaiian species complexes: Montipora dilatata/flabellata/turgescens & M. patula/verrilli.

    Directory of Open Access Journals (Sweden)

    Zac H Forsman

    Full Text Available M. dilatata, M. flabellata, and M. patula and 80 other scleractinian corals were petitioned to be listed under the US Endangered Species Act (ESA, which would have major conservation implications. One of the difficulties with this evaluation is that reproductive boundaries between morphologically defined coral species are often permeable, and morphology can be wildly variable. We examined genetic and morphological variation in Hawaiian Montipora with a suite of molecular markers (mitochondrial: COI, CR, Cyt-B, 16S, ATP6; nuclear: ATPsβ, ITS and microscopic skeletal measurements. Mitochondrial markers and the ITS region revealed four distinct clades: I M. patula/M. verrilli, II M. cf. incrassata, III M. capitata, IV M. dilatata/M. flabellata/M. cf. turgescens. These clades are likely to occur outside of Hawai'i according to mitochondrial control region haplotypes from previous studies. The ATPsβ intron data showed a pattern often interpreted as resulting from hybridization and introgression; however, incomplete lineage sorting may be more likely since the multicopy nuclear ITS region was consistent with the mitochondrial data. Furthermore, principal components analysis (PCA of skeletal microstructure was concordant with the mitochondrial clades, while nominal taxa overlapped. The size and shape of verrucae or papillae contributed most to identifying groups, while colony-level morphology was highly variable. It is not yet clear if these species complexes represent population-level variation or incipient speciation (CA<1MYA, two alternatives that have very different conservation implications. This study highlights the difficulty in understanding the scale of genetic and morphological variation that corresponds to species as opposed to population-level variation, information that is essential for conservation and for understanding coral biodiversity.

  3. Complex interactions of the Eastern and Western Slavic populations with other European groups as revealed by mitochondrial DNA analysis.

    Science.gov (United States)

    Grzybowski, Tomasz; Malyarchuk, Boris A; Derenko, Miroslava V; Perkova, Maria A; Bednarek, Jarosław; Woźniak, Marcin

    2007-06-01

    Mitochondrial DNA sequence variation was examined by the control region sequencing (HVS I and HVS II) and RFLP analysis of haplogroup-diagnostic coding region sites in 570 individuals from four regional populations of Poles and two Russian groups from northwestern part of the country. Additionally, sequences of complete mitochondrial genomes representing K1a1b1a subclade in Polish and Polish Roma populations have been determined. Haplogroup frequency patterns revealed in Poles and Russians are similar to those characteristic of other Europeans. However, there are several features of Slavic mtDNA pools seen on the level of regional populations which are helpful in the understanding of complex interactions of the Eastern and Western Slavic populations with other European groups. One of the most important is the presence of subhaplogroups U5b1b1, D5, Z1 and U8a with simultaneous scarcity of haplogroup K in populations of northwestern Russia suggesting the participation of Finno-Ugrian tribes in the formation of mtDNA pools of Russians from this region. The results of genetic structure analyses suggest that Russians from Velikii Novgorod area (northwestern Russia) and Poles from Suwalszczyzna (northeastern Poland) differ from all remaining Polish and Russian samples. Simultaneously, northwestern Russians and northeastern Poles bear some similarities to Baltic (Latvians) and Finno-Ugrian groups (Estonians) of northeastern Europe, especially on the level of U5 haplogroup frequencies. The occurrence of K1a1b1a subcluster in Poles and Polish Roma is one of the first direct proofs of the presence of Ashkenazi-specific mtDNA lineages in non-Jewish European populations.

  4. Deep sequencing of the murine Igh repertoire reveals complex regulation of non-random V gene rearrangement frequencies

    Science.gov (United States)

    Choi, Nancy M.; Loguercio, Salvatore; Verma-Gaur, Jiyoti; Degner, Stephanie C.; Torkamani, Ali; Su, Andrew I.; Oltz, Eugene M.; Artyomov, Maxim; Feeney, Ann J.

    2013-01-01

    A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5′-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that being positioned near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have higher levels of histone marks and non-coding RNA, which may compensate for their poorer RSSs and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for each of the four domains that comprise this locus. PMID:23898036

  5. Complex mean circulation over the inner shelf south of Martha's Vineyard revealed by observations and a high-resolution model

    Science.gov (United States)

    Ganju, Neil K.; Lentz, Steven J.; Kirincich, Anthony R.; Farrar, J. Thomas

    2011-01-01

    Inner-shelf circulation is governed by the interaction between tides, baroclinic forcing, winds, waves, and frictional losses; the mean circulation ultimately governs exchange between the coast and ocean. In some cases, oscillatory tidal currents interact with bathymetric features to generate a tidally rectified flow. Recent observational and modeling efforts in an overlapping domain centered on the Martha's Vineyard Coastal Observatory (MVCO) provided an opportunity to investigate the spatial and temporal complexity of circulation on the inner shelf. ADCP and surface radar observations revealed a mean circulation pattern that was highly variable in the alongshore and cross-shore directions. Nested modeling incrementally improved representation of the mean circulation as grid resolution increased and indicated tidal rectification as the generation mechanism of a counter-clockwise gyre near the MVCO. The loss of model skill with decreasing resolution is attributed to insufficient representation of the bathymetric gradients (Δh/h), which is important for representing nonlinear interactions between currents and bathymetry. The modeled momentum balance was characterized by large spatial variability of the pressure gradient and horizontal advection terms over short distances, suggesting that observed inner-shelf momentum balances may be confounded. Given the available observational and modeling data, this work defines the spatially variable mean circulation and its formation mechanism—tidal rectification—and illustrates the importance of model resolution for resolving circulation and constituent exchange near the coast. The results of this study have implications for future observational and modeling studies near the MVCO and other inner-shelf locations with alongshore bathymetric variability.

  6. Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities.

    Science.gov (United States)

    Starchenka, S; Bell, A J; Mwange, J; Skinner, M A; Heath, M D

    2017-01-01

    Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass. HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model. Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract. The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an 'immunodominant' Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept

  7. Reinvestigation of aminoacyl-tRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex.

    Directory of Open Access Journals (Sweden)

    Kyutae Kim

    Full Text Available Twenty different aminoacyl-tRNA synthetases (ARSs link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS, together with three ARS-interacting multifunctional proteins (AIMPs, are currently known to assemble the multi-synthetase complex (MSC. However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2 was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.

  8. Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior

    Science.gov (United States)

    Wilson-Kubalek, Elizabeth M.; Cheeseman, Iain M.; Milligan, Ronald A.

    2016-01-01

    During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore–microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore–microtubule attachments. Here we compare the binding of the Caenorhabditis elegans and human Ndc80 complexes to microtubules at high resolution using cryo–electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas the C. elegans Ndc80 complex binds more tightly to β-tubulin. In addition, the C. elegans Ndc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore–microtubule interactions. PMID:26941333

  9. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  10. Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Vester-Christensen, Malene Bech; Jensen, Johanne M.

    2015-01-01

    , and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding...

  11. A developmental framework for complex plasmodesmata formation revealed by large-scale imaging of the Arabidopsis leaf epidermis.

    Science.gov (United States)

    Fitzgibbon, Jessica; Beck, Martina; Zhou, Ji; Faulkner, Christine; Robatzek, Silke; Oparka, Karl

    2013-01-01

    Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink-source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.

  12. Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain.

    Science.gov (United States)

    Taurino, Federica; Stanca, Eleonora; Siculella, Luisa; Trentadue, Raffaella; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2012-04-18

    Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.

  13. All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes.

    Science.gov (United States)

    Kokhan, Oleksandr; Shinkarev, Vladimir P

    2011-02-02

    Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q(i) site of the bacterial and bovine bc(1) complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q(i) pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q(i) pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc(1) complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc(1) complex and only rarely in the bovine bc(1) complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc(1) complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc(1) complex.

  14. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging*♦

    Science.gov (United States)

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M.; Johnson, Aaron M.; Ren, Xiaojun

    2015-01-01

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. PMID:26381410

  15. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    Science.gov (United States)

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

  16. Mechanism of Formation of Copper(II) Chloro Complexes Revealed by Transient Absorption Spectroscopy and DFT/TDDFT Calculations.

    Science.gov (United States)

    Mereshchenko, Andrey S; Olshin, Pavel K; Karabaeva, Kanykey E; Panov, Maxim S; Wilson, R Marshall; Kochemirovsky, Vladimir A; Skripkin, Mikhail Yu; Tveryanovich, Yury S; Tarnovsky, Alexander N

    2015-07-16

    Copper(II) complexes are extremely labile with typical ligand exchange rate constants on the order of 10(6)-10(9) M(-1) s(-1). As a result, it is often difficult to identify the actual formation mechanism of these complexes. In this work, using UV-vis transient absorption when probing in a broad time range (20 ps to 8 μs) in conjunction with DFT/TDDFT calculations, we studied the dynamics and underlying reaction mechanisms of the formation of extremely labile copper(II) CuCl4(2-) chloro complexes from copper(II) CuCl3(-) trichloro complexes and chloride ions. These two species, produced via photochemical dissociation of CuCl4(2-) upon 420 nm excitation into the ligand-to-metal-charge-transfer electronic state, are found to recombine into parent complexes with bimolecular rate constants of (9.0 ± 0.1) × 10(7) and (5.3 ± 0.4) × 10(8) M(-1) s(-1) in acetonitrile and dichloromethane, respectively. In dichloromethane, recombination occurs via a simple one-step addition. In acetonitrile, where [CuCl3](-) reacts with the solvent to form a [CuCl3CH3CN](-) complex in less than 20 ps, recombination takes place via ligand exchange described by the associative interchange mechanism that involves a [CuCl4CH3CN](2-) intermediate. In both solvents, the recombination reaction is potential energy controlled.

  17. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.

    Science.gov (United States)

    Smits, Arne H; Jansen, Pascal W T C; Poser, Ina; Hyman, Anthony A; Vermeulen, Michiel

    2013-01-07

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

  18. Ion mobility-mass spectrometry of charge-reduced protein complexes reveals general trends in the collisional ejection of compact subunits.

    Science.gov (United States)

    Bornschein, Russell E; Ruotolo, Brandon T

    2015-10-21

    Multiprotein complexes have been shown to play critical roles across a wide range of cellular functions, but most probes of protein quaternary structure are limited in their ability to analyze complex mixtures and polydisperse structures using small amounts of total protein. Ion mobility-mass spectrometry offers a solution to many of these challenges, but relies upon gas-phase measurements of intact multiprotein complexes, subcomplexes, and subunits that correlate well with solution structures. The greatest bottleneck in such workflows is the generation of representative subcomplexes and subunits. Collisional activation of complexes can act to produce product ions reflective of protein complex composition, but such product ions are typically challenging to interpret in terms of their relationship to solution structure due to their typically string-like conformations following activation and subsequent dissociation. Here, we used ion-ion chemistry to perform a broad survey of the gas-phase dissociation of charge-reduced protein complex ions, revealing general trends associated with the collisional ejection of compact, rather than unfolded, protein subunits. Furthermore, we also discover peptide and co-factor dissociation channels that dominate the product ion populations generated for such charge reduced complexes. We assess both sets of observations and discuss general principles that can be extended to the analysis of protein complex ions having unknown structures.

  19. Introgressive hybridization and the evolutionary history of the herring gull complex revealed by mitochondrial and nuclear DNA

    Directory of Open Access Journals (Sweden)

    Zhang Jun

    2010-11-01

    Full Text Available Abstract Background Based on extensive mitochondrial DNA (mtDNA sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from sufficient to obtain a more accurate and detailed insight into the demographic processes that underlie speciation of this complex, and that extensive autosomal genetic analysis was warranted. Results For this reason, the present study focuses on the reconstruction of the phylogeographic history of a limited number of gull species by means of a combined approach of mtDNA sequence data and 230 autosomal amplified fragment length polymorphism (AFLP loci. At the species level, the mtDNA and AFLP genetic data were largely congruent. Not only for argentatus and hyperboreus, but also among a third species, great black-backed gull (L. marinus we observed two distinct groups of mtDNA sequence haplotypes. Based on the AFLP data we were also able to detect distinct genetic subgroups among the various argentatus, hyperboreus, and marinus populations, supporting our initial hypothesis that complex demographic scenario's underlie speciation in the herring gull complex. Conclusions We present evidence that for each of these three biphyletic gull species, extensive mtDNA introgression could have taken place among the various geographically distinct subpopulations, or even among current species. Moreover, based on a large number of autosomal AFLP loci, we found evidence for distinct and complex demographic scenario's for each of the three species we studied. A more refined insight into the exact phylogeographic history within the herring gull complex is still impossible, and requires

  20. Complex

    African Journals Online (AJOL)

    CLEMENT O BEWAJI

    Schiff bases and their complex compounds have been studied for their .... establishing coordination of the N–(2 – hydroxybenzyl) - L - α - valine Schiff base ..... (1967); “Spectrophotometric Identification of Organic Compounds”, Willey, New.

  1. Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

    Directory of Open Access Journals (Sweden)

    Sepil Irem

    2012-05-01

    Full Text Available Abstract Background The critical role of Major Histocompatibility Complex (Mhc genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help

  2. Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment

    Science.gov (United States)

    Byron, Adam; Humphries, Jonathan D; Craig, Sue E; Knight, David; Humphries, Martin J

    2012-01-01

    Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events. PMID:22623428

  3. Quantitative imaging reveals real-time Pou5f3–Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

    Science.gov (United States)

    Perez-Camps, Mireia; Tian, Jing; Chng, Serene C; Sem, Kai Pin; Sudhaharan, Thankiah; Teh, Cathleen; Wachsmuth, Malte; Korzh, Vladimir; Ahmed, Sohail; Reversade, Bruno

    2016-01-01

    Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3–Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3–Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3–Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis. DOI: http://dx.doi.org/10.7554/eLife.11475.001 PMID:27684073

  4. Intracortical Microstimulation Maps of Motor, Somatosensory, and Posterior Parietal Cortex in Tree Shrews (Tupaia belangeri) Reveal Complex Movement Representations.

    Science.gov (United States)

    Baldwin, Mary K L; Cooke, Dylan F; Krubitzer, Leah

    2016-01-11

    Long-train intracortical microstimulation (LT-ICMS) is a popular method for studying the organization of motor and posterior parietal cortex (PPC) in mammals. In primates, LT-ICMS evokes both multijoint and multiple-body-part movements in primary motor, premotor, and PPC. In rodents, LT-ICMS evokes complex movements of a single limb in motor cortex. Unfortunately, very little is known about motor/PPC organization in other mammals. Tree shrews are closely related to both primates and rodents and could provide insights into the evolution of complex movement domains in primates. The present study investigated the extent of cortex in which movements could be evoked with ICMS and the characteristics of movements elicited using both short train (ST) and LT-ICMS in tree shrews. We demonstrate that LT-ICMS and ST-ICMS maps are similar, with the movements elicited with ST-ICMS being truncated versions of those elicited with LT-ICMS. In addition, LT-ICMS-evoked complex movements within motor cortex similar to those in rodents. More complex movements involving multiple body parts such as the hand and mouth were also elicited in motor cortex and PPC, as in primates. Our results suggest that complex movement networks present in PPC and motor cortex were present in mammals prior to the emergence of primates.

  5. Heterogeneity in optical properties of near white-light emissive europium complex species revealed by spectroscopy of single nanoaggregates

    Science.gov (United States)

    Irfanullah, Mir; Sharma, Dharmendar Kumar; Chulliyil, Ramya; Layek, Arunasish; De, Suman; Chowdhury, Arindam

    2017-01-01

    Photoluminescence microscopy has been used to interrogate individual nanoaggregates (NAs) of visible light excitable Eu(III)-complex species with 9-hydroxyphenalenone which we found to emit near-white light in methanol. In the solid state however, NAs display diverse emission spectra due to varied sensitization efficiencies, and thereby exhibit a wide range of emission colours. Heterogeneity in sensitization efficiency and asymmetry ratios for Eu-emission is intriguing because all measurable photoluminescence parameters are expected to average out over large number of Eu-complex species which constitute NAs, and suggests the existence of relatively few yet efficient Eu3+ radiative trap centres of varied asymmetry within NAs.

  6. Earlier timbre processing of instrumental tones compared to equally complex spectrally rotated sounds as revealed by the mismatch negativity.

    Science.gov (United States)

    Christmann, Corinna A; Lachmann, Thomas; Berti, Stefan

    2014-10-03

    Harmonically rich sounds have been shown to be processed more efficiently by the human brain compared to single sinusoidal tones. To control for stimulus complexity as a potentially confounding factor, tones and equally complex spectrally rotated sounds, have been used in the present study to investigate the role of the overtone series in sensory auditory processing in non-musicians. Timbre differences in instrumental tones with equal pitch elicited a MMN which was earlier compared to that elicited by the spectrally rotated sounds, indicating that harmonically rich tones are processed faster compared to non-musical sounds without an overtone series, even when pitch is not the relevant information.

  7. Linking Complex Problem Solving and General Mental Ability to Career Advancement: Does a Transversal Skill Reveal Incremental Predictive Validity?

    Science.gov (United States)

    Mainert, Jakob; Kretzschmar, André; Neubert, Jonas C.; Greiff, Samuel

    2015-01-01

    Transversal skills, such as complex problem solving (CPS) are viewed as central twenty-first-century skills. Recent empirical findings have already supported the importance of CPS for early academic advancement. We wanted to determine whether CPS could also contribute to the understanding of career advancement later in life. Towards this end, we…

  8. A single-molecule view of DNA replication : the dynamic nature of multi-protein complexes revealed

    NARCIS (Netherlands)

    Geertsema, Hylkje J.; van Oijen, Antoine M.; Chiu, Wah; Wagner, Gerhard

    2013-01-01

    Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multiprotein complexes that support multiple enzymatic activities. In

  9. Untangling a species complex of arid zone grasses (Triodia) reveals patterns congruent with co-occurring animals.

    Science.gov (United States)

    Anderson, Benjamin M; Barrett, Matthew D; Krauss, Siegfried L; Thiele, Kevin

    2016-08-01

    The vast Australian arid zone formed over the last 15million years, and gradual aridification as well as more extreme Pliocene and Pleistocene climate shifts have impacted the evolution of its biota. Understanding the evolutionary history of groups of organisms or regional biotas such as the Australian arid biota requires clear delimitation of the units of biodiversity (taxa). Here we integrate evidence from nuclear (ETS and ITS) and chloroplast (rps16-trnK spacer) regions and morphology to clarify taxonomic boundaries in a species complex of Australian hummock grasses (Triodia) to better understand the evolution of Australian arid zone plants and to evaluate congruence in distribution patterns with co-occurring organisms. We find evidence for multiple new taxa in the T. basedowii species complex, but also incongruence between data sets and indications of hybridization that complicate delimitation. We find that the T. basedowii complex has high lineage diversity and endemism in the biologically important Pilbara region of Western Australia, consistent with the region acting as a refugium. Taxa show strong geographic structure in the Pilbara, congruent with recent work on co-occurring animals and suggesting common evolutionary drivers across the biota. Our findings confirm recognition of the Pilbara as an important centre of biodiversity in the Australian arid zone, and provide a basis for future taxonomic revision of the T. basedowii complex and more detailed study of its evolutionary history and that of arid Australia.

  10. Revealing and tuning the core, structure, properties and function of polymer micelles with lanthanide-coordination complexes

    NARCIS (Netherlands)

    Wang, J.; Groeneveld, A.; Oikonomou, M.E.; Prusova, A.; As, van H.; Lent, van J.W.M.; Velders, A.H.

    2016-01-01

    Controlling self-assembly processes is of great interest in various fields where multifunctional and tunable materials are designed. We here present the versatility of lanthanide-complex-based micelles (Ln-C3Ms) with tunable coordination structures and corresponding functions (e.g. luminescence and

  11. Letter: Variable and complex food web structures revealed by exploring missing trophic links between birds and biofilm

    NARCIS (Netherlands)

    Kuwae, T.; Miyoshi, E.; Hosokawa, S.; Amano, T.; Moriya, T.; Kondoh, M.; Ydenberg, R.C.; Elner, R.W.

    2012-01-01

    Food webs are comprised of a network of trophic interactions and are essential to elucidating ecosystem processes and functions. However, the presence of unknown, but critical networks hampers understanding of complex and dynamic food webs in nature. Here, we empirically demonstrate a missing link,

  12. A sensitive LC-ESI-Q-TOF-MS method reveals novel phytosiderophores and phytosiderophore-iron complexes in barley.

    Science.gov (United States)

    Tsednee, Munkhtsetseg; Mak, Yit-Wai; Chen, Yet-Ran; Yeh, Kuo-Chen

    2012-09-01

    The direct analysis of phytosiderophores (PSs) and their metal complexes in plants is critical to understanding the biological functions of different PSs. Here we report on a rapid and highly sensitive liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) method for the direct and simultaneous determination of free PSs and their ferric complexes in plants. In addition to previously reported PSs--deoxymugineic acid (DMA), mugineic acid (MA) and epihydroxymugineic acid (epi-HMA)--two more PSs, avenic acid (AVA) and hydroxyavenic acid (HAVA), were identified by this method in roots of Hordeum vulgare cv Himalaya and in root exudates under iron (Fe) deficiency. The two identified PSs could be responsible for Fe acquisition under Fe deficiency because of their relative abundance and ability to form ferric complexes in secreted root exudates. This LC-ESI-Q-TOF-MS method greatly facilitates the identification of free PSs and PS-Fe complexes in one plant sample. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  13. A systematic review of cost-effectiveness analyses of complex wound interventions reveals optimal treatments for specific wound types.

    Science.gov (United States)

    Tricco, Andrea C; Cogo, Elise; Isaranuwatchai, Wanrudee; Khan, Paul A; Sanmugalingham, Geetha; Antony, Jesmin; Hoch, Jeffrey S; Straus, Sharon E

    2015-04-22

    Complex wounds present a substantial economic burden on healthcare systems, costing billions of dollars annually in North America alone. The prevalence of complex wounds is a significant patient and societal healthcare concern and cost-effective wound care management remains unclear. This article summarizes the cost-effectiveness of interventions for complex wound care through a systematic review of the evidence base. We searched multiple databases (MEDLINE, EMBASE, Cochrane Library) for cost-effectiveness studies that examined adults treated for complex wounds. Two reviewers independently screened the literature, abstracted data from full-text articles, and assessed methodological quality using the Drummond 10-item methodological quality tool. Incremental cost-effectiveness ratios were reported, or, if not reported, calculated and converted to United States Dollars for the year 2013. Overall, 59 cost-effectiveness analyses were included; 71% (42 out of 59) of the included studies scored 8 or more points on the Drummond 10-item checklist tool. Based on these, 22 interventions were found to be more effective and less costly (i.e., dominant) compared to the study comparators: 9 for diabetic ulcers, 8 for venous ulcers, 3 for pressure ulcers, 1 for mixed venous and venous/arterial ulcers, and 1 for mixed complex wound types. Our results can be used by decision-makers in maximizing the deployment of clinically effective and resource efficient wound care interventions. Our analysis also highlights specific treatments that are not cost-effective, thereby indicating areas of resource savings. Please see related article: http://dx.doi.org/10.1186/s12916-015-0288-5.

  14. High-resolution crystal structure of Streptococcus pyogenes β-NAD{sup +} glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Ji Young; An, Doo Ri; Yoon, Hye-Jin [Seoul National University, Seoul 151-747 (Korea, Republic of); Kim, Hyoun Sook [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-742 (Korea, Republic of); Lee, Sang Jae [Seoul National University, Seoul 151-742 (Korea, Republic of); Im, Ha Na; Jang, Jun Young [Seoul National University, Seoul 151-747 (Korea, Republic of); Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-747 (Korea, Republic of)

    2013-11-01

    The crystal structure of the complex between the C-terminal domain of Streptococcus pyogenes β-NAD{sup +} glycohydrolase and an endogenous inhibitor for SPN was determined at 1.70 Å. It reveals that the interface between the two proteins is highly rich in water molecules. One of the virulence factors produced by Streptococcus pyogenes is β-NAD{sup +} glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38–451) and the full-length IFS (residues 1–161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPN{sub ct}–IFS complex, which consists of the SPN C-terminal domain (SPN{sub ct}; residues 193–451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPN{sub ct} and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope.

  15. High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design.

    Science.gov (United States)

    Sippel, Katherine H; Robbins, Arthur H; Domsic, John; Genis, Caroli; Agbandje-McKenna, Mavis; McKenna, Robert

    2009-10-01

    The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1 A resolution and refined to an R(cryst) of 11.2% and an R(free) of 14.7%. As observed in previous CA II-inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed.

  16. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry

    DEFF Research Database (Denmark)

    Chen, Zhuo Angel; Jawhari, Anass; Fischer, Lutz

    2010-01-01

    to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA....... The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws...

  17. Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy

    Science.gov (United States)

    Rätsep, Margus; Pajusalu, Mihkel; Linnanto, Juha Matti; Freiberg, Arvi

    2014-10-01

    We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments.

  18. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  19. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.

    Science.gov (United States)

    Chamberlain, Philip P; Lopez-Girona, Antonia; Miller, Karen; Carmel, Gilles; Pagarigan, Barbra; Chie-Leon, Barbara; Rychak, Emily; Corral, Laura G; Ren, Yan J; Wang, Maria; Riley, Mariko; Delker, Silvia L; Ito, Takumi; Ando, Hideki; Mori, Tomoyuki; Hirano, Yoshinori; Handa, Hiroshi; Hakoshima, Toshio; Daniel, Thomas O; Cathers, Brian E

    2014-09-01

    The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

  20. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  1. Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Rätsep, Margus, E-mail: margus.ratsep@ut.ee; Pajusalu, Mihkel, E-mail: mihkel.pajusalu@ut.ee; Linnanto, Juha Matti, E-mail: juha.matti.linnanto@ut.ee [Institute of Physics, University of Tartu, Riia 142, 51014 Tartu (Estonia); Freiberg, Arvi, E-mail: arvi.freiberg@ut.ee [Institute of Physics, University of Tartu, Riia 142, 51014 Tartu, Estonia and Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu (Estonia)

    2014-10-21

    We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments.

  2. Crystal structure of a PCP/Sfp complex reveals the structural basis for carrier protein posttranslational modification.

    Science.gov (United States)

    Tufar, Peter; Rahighi, Simin; Kraas, Femke I; Kirchner, Donata K; Löhr, Frank; Henrich, Erik; Köpke, Jürgen; Dikic, Ivan; Güntert, Peter; Marahiel, Mohamed A; Dötsch, Volker

    2014-04-24

    Phosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier protein's second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life.

  3. Species-richness of the Anopheles annulipes Complex (Diptera: Culicidae) Revealed by Tree and Model-Based Allozyme Clustering Analyses

    Science.gov (United States)

    2007-01-01

    and systematics ofAnopheles: insights from a molecular phy· logeny of Australasian mosquitoes. Molecular Phylogenetics and Evolution 9: 262-275. Foley... systematics and pop- ulation studies. Sydney: Academic Press. Roscnberg NA, Burke T, Elo K, Feldman MW, Freidlin PJ, Groenen MAM, Hillel J, Maki·Taniia...of molecular-functional variation patterns among butterfly species complexes IColias: Lepidoptera , Pieridae). Molecular Ecology 12: 1265-1275. © 2007

  4. A Comprehensive Image-based Phenomic Analysis Reveals the Complex Genetic Architecture of Shoot Growth Dynamics in Rice (Oryza sativa).

    Science.gov (United States)

    Campbell, Malachy T; Du, Qian; Liu, Kan; Brien, Chris J; Berger, Bettina; Zhang, Chi; Walia, Harkamal

    2017-07-01

    Early vigor is an important trait for many rice ( L.)-growing environments. However, genetic characterization and improvement for early vigor is hindered by the temporal nature of the trait and strong genotype × environment effects. We explored the genetic architecture of shoot growth dynamics during the early and active tillering stages by applying a functional modeling and genomewide association (GWAS) mapping approach on a diversity panel of ∼360 rice accessions. Multiple loci with small effects on shoot growth trajectory were identified, indicating a complex polygenic architecture. Natural variation for shoot growth dynamics was assessed in a subset of 31 accessions using RNA sequencing and hormone quantification. These analyses yielded a gibberellic acid (GA) catabolic gene, , which could influence GA levels to regulate vigor in the early tillering stage. Given the complex genetic architecture of shoot growth dynamics, the potential of genomic selection (GS) for improving early vigor was explored using all 36,901 single-nucleotide polymorphisms (SNPs) as well as several subsets of the most significant SNPs from GWAS. Shoot growth trajectories could be predicted with reasonable accuracy using the 50 most significant SNPs from GWAS (0.37-0.53); however, the accuracy of prediction was improved by including more markers, which indicates that GS may be an effective strategy for improving shoot growth dynamics during the vegetative growth stage. This study provides insights into the complex genetic architecture and molecular mechanisms underlying early shoot growth dynamics and provides a foundation for improving this complex trait in rice. Copyright © 2017 Crop Science Society of America.

  5. High resolution digital elevation modelling from TLS and UAV campaign reveals structural complexity at the 2014 Holuhraun eruption site

    Science.gov (United States)

    Müller, Daniel; Walter, Thomas R.; Titt, Tanja; Schöpa, Anne; Tumi Gudmundsson, Magnus; Dürig, Tobi

    2017-04-01

    Fissure eruptions are commonly linked to magma dikes at depth, associated with deformation that is described by subsidence and lateral widening at the surface. The structure formation associated with such fissure eruptions, however, is barely preserved in nature because of the rapid erosion and/or difficult access to these areas, which is why, so far, normal fault displacements are commonly assumed for this type of fractures. At the 2014 Holuhraun eruption sites, the largest fissure eruption in Iceland since almost two centuries, evidence is increasing that the developing structures are related to pre-existing topography, reactivation of earlier fractures and possible complexity in the opening mode of the dike. In an attempt to investigate the Holuhraun structures in greater detail, a fieldwork mapping project combining terrestrial laser scanning (TLS) and unmanned aerial vehicle (UAV) based aerophoto analysis was realized. From this data, we generated a locally high resolution digital elevation model and a structural map that allow for identification of kinematic indicators and assessing senses of fault opening, strike-slip movements, and complexities in fracture pathways. We identified fracture curvatures, step-overs and en-echelon type structures, and measured strike directions for single fault segments including the amount of opening and opening angles. We conjecture that local complexities in the fracture paths and fracture geometries are closely related to pre-existing geometric and mechanical heterogeneities. Moreover, we identified local changes in fracture trends and offsets close to eruption sites, which are possibly associated with geometrical changes in the feeding dike itself. Results have important implications for the development of surface structures at fissure eruption sites and underline that the structural memory is a very important factor in understanding the complexities of local fault structures above dike intrusions.

  6. Rethinking the longitudinal stream temperature paradigm: region-wide comparison of thermal infrared imagery reveals unexpected complexity of river temperatures

    Science.gov (United States)

    Fullerton, Aimee H.; Torgersen, Christian; Lawler, Joshua J.; Faux, Russell N.; Steel, E. Ashley; Beechie, Timothy J.; Ebersole, Joseph L.; Leibowitz, Scott J.

    2015-01-01

    Prevailing theory suggests that stream temperature warms asymptotically in a downstream direction, beginning at the temperature of the source in the headwaters and leveling off downstream as it converges to match meteorological conditions. However, there have been few empirical examples of longitudinal patterns of temperature in large rivers due to a paucity of data. We constructed longitudinal thermal profiles (temperature versus distance) for 53 rivers in the Pacific Northwest (USA) using an extensive dataset of remotely sensed summertime river temperatures and classified each profile into one of five patterns of downstream warming: asymptotic (increasing then flattening), linear (increasing steadily), uniform (not changing), parabolic (increasing then decreasing), or complex (not fitting other classes). We evaluated (1) how frequently profiles warmed asymptotically downstream as expected, and (2) whether relationships between river temperature and common hydroclimatic variables differed by profile class. We found considerable diversity in profile shape, with 47% of rivers warming asymptotically, and 53% having alternative profile shapes. Water temperature did not warm substantially over the course of the river for coastal parabolic and uniform profiles, and for some linear and complex profiles. Profile classes showed no clear geographical trends. The degree of correlation between river temperature and hydroclimatic variables differed among profile classes, but there was overlap among classes. Water temperature in rivers with asymptotic or parabolic profiles was positively correlated with August air temperature, tributary temperature and velocity, and negatively correlated with elevation, August precipitation, gradient, and distance upstream. Conversely, associations were less apparent in rivers with linear, uniform, or complex profiles. Factors contributing to the unique shape of parabolic profiles differed for coastal and inland rivers, where downstream cooling

  7. Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of 227 antibody-antigen complexes.

    Science.gov (United States)

    Robin, Gautier; Sato, Yoshiteru; Desplancq, Dominique; Rochel, Natacha; Weiss, Etienne; Martineau, Pierre

    2014-11-11

    Antibody molecules are able to recognize any antigen with high affinity and specificity. To get insight into the molecular diversity at the source of this functional diversity, we compiled and analyzed a non-redundant aligned collection of 227 structures of antibody-antigen complexes. Free energy of binding of all the residue side chains was quantified by computational alanine scanning, allowing the first large-scale quantitative description of antibody paratopes. This demonstrated that as few as 8 residues among 30 key positions are sufficient to explain 80% of the binding free energy in most complexes. At these positions, the residue distribution is not only different from that of other surface residues but also dependent on the role played by the side chain in the interaction, residues participating in the binding energy being mainly aromatic residues, and Gly or Ser otherwise. To question the generality of these binding characteristics, we isolated an antibody fragment by phage display using a biased synthetic repertoire with only two diversified complementarity-determining regions and solved its structure in complex with its antigen. Despite this restricted diversity, the structure demonstrated that all complementarity-determining regions were involved in the interaction with the antigen and that the rules derived from the natural antibody repertoire apply to this synthetic binder, thus demonstrating the robustness and universality of our results. Copyright © 2014. Published by Elsevier Ltd.

  8. Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design.

    Science.gov (United States)

    Wang, Ling; Althoff, Eric A; Bolduc, Jill; Jiang, Lin; Moody, James; Lassila, Jonathan K; Giger, Lars; Hilvert, Donald; Stoddard, Barry; Baker, David

    2012-01-20

    We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme-inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl-substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.

  9. Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase.

    Science.gov (United States)

    Mesquita, Ana; Tábara, Luis C; Martinez-Costa, Oscar; Santos-Rodrigo, Natalia; Vincent, Olivier; Escalante, Ricardo

    2015-08-01

    The network of protein-protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.

  10. Excitation relaxation dynamics and energy transfer in pigment-protein complexes of a dinoflagellate, revealed by ultrafast fluorescence spectroscopy.

    Science.gov (United States)

    Tanaka, Kazunori; Iida, Satoko; Takaichi, Shinichi; Mimuro, Mamoru; Murakami, Akio; Akimoto, Seiji

    2016-12-01

    Photosynthetic light-harvesting complexes, found in aquatic photosynthetic organisms, contain a variety of carotenoids and chlorophylls. Most of the photosynthetic dinoflagellates possess two types of light-harvesting antenna complexes: peridinin (Peri)-chlorophyll (Chl) a/c-protein, as an intrinsic thylakoid membrane complex protein (iPCP), and water-soluble Peri-Chl a-protein, as an extrinsic membrane protein (sPCP) on the inner surface of the thylakoid. Peri is a unique carotenoid that has eight C=C bonds and one C=O bond, which results in a characteristic absorption band in the green wavelength region. In the present study, excitation relaxation dynamics of Peri in solution and excitation energy transfer processes of sPCP and the thylakoid membranes, prepared from the photosynthetic dinoflagellate, Symbiodinium sp., are investigated by ultrafast time-resolved fluorescence spectroscopy. We found that Peri-to-Chl a energy transfer occurs via the Peri S1 state with a time constant of 1.5 ps or 400 fs in sPCP or iPCP, respectively, and that Chl c-to-Chl a energy transfer occurs in the time regions of 350-400 fs and 1.8-2.6 ps.

  11. Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

    OpenAIRE

    2010-01-01

    Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2 kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of t...

  12. Solution structure of the dimerization domain of the eukaryotic stalk P1/P2 complex reveals the structural organization of eukaryotic stalk complex.

    Science.gov (United States)

    Lee, Ka-Ming; Yu, Conny Wing-Heng; Chiu, Teddy Yu-Hin; Sze, Kong-Hung; Shaw, Pang-Chui; Wong, Kam-Bo

    2012-04-01

    The lateral ribosomal stalk is responsible for the kingdom-specific binding of translation factors and activation of GTP hydrolysis during protein synthesis. The eukaryotic stalk is composed of three acidic ribosomal proteins P0, P1 and P2. P0 binds two copies of P1/P2 hetero-dimers to form a pentameric P-complex. The structure of the eukaryotic stalk is currently not known. To provide a better understanding on the structural organization of eukaryotic stalk, we have determined the solution structure of the N-terminal dimerization domain (NTD) of P1/P2 hetero-dimer. Helix-1, -2 and -4 from each of the NTD-P1 and NTD-P2 form the dimeric interface that buries 2200 A(2) of solvent accessible surface area. In contrast to the symmetric P2 homo-dimer, P1/P2 hetero-dimer is asymmetric. Three conserved hydrophobic residues on the surface of NTD-P1 are replaced by charged residues in NTD-P2. Moreover, NTD-P1 has an extra turn in helix-1, which forms extensive intermolecular interactions with helix-1 and -4 of NTD-P2. Truncation of this extra turn of P1 abolished the formation of P1/P2 hetero-dimer. Systematic truncation studies suggest that P0 contains two spine-helices that each binds one copy of P1/P2 hetero-dimer. Modeling studies suggest that a large hydrophobic cavity, which can accommodate the loop between the spine-helices of P0, can be found on NTD-P1 but not on NTD-P2 when the helix-4 adopts an 'open' conformation. Based on the asymmetric properties of NTD-P1/NTD-P2, a structural model of the eukaryotic P-complex with P2/P1:P1/P2 topology is proposed.

  13. Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains.

    Science.gov (United States)

    Clark, Howard W; Mackay, Rose-Marie; Deadman, Mary E; Hood, Derek W; Madsen, Jens; Moxon, E Richard; Townsend, J Paul; Reid, Kenneth B M; Ahmed, Abdul; Shaw, Amy J; Greenhough, Trevor J; Shrive, Annette K

    2016-05-01

    The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. Using Haemophilus influenzae Eagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325. Combined with enzyme-linked immunosorbent assays (ELISAs) and fluorescence-activated cell sorter (FACS) binding analyses, our results show that extended LPS structures previously thought to be targets for collectins are important in shielding the more vulnerable sites in the LPS core, revealing a mechanism by which pathogens with complex LPS extensions efficiently evade a first-line mucosal innate immune defense. The structure also reveals for the first time the dominant form of anhydro-Kdo.

  14. Expression profiling of major histocompatibility and natural killer complex genes reveals candidates for controlling risk of graft versus host disease.

    Directory of Open Access Journals (Sweden)

    Peter Novota

    Full Text Available BACKGROUND: The major histocompatibility complex (MHC is the most important genomic region that contributes to the risk of graft versus host disease (GVHD after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. METHODOLOGY/PRINCIPAL FINDINGS: To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR, to analyze the expression of MHC, natural killer complex (NKC, and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. CONCLUSIONS/SIGNIFICANCE: We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

  15. Expression profiling of major histocompatibility and natural killer complex genes reveals candidates for controlling risk of graft versus host disease.

    Science.gov (United States)

    Novota, Peter; Zinöcker, Severin; Norden, Jean; Wang, Xiao Nong; Sviland, Lisbet; Opitz, Lennart; Salinas-Riester, Gabriela; Rolstad, Bent; Dickinson, Anne M; Walter, Lutz; Dressel, Ralf

    2011-01-28

    The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

  16. Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

    OpenAIRE

    2010-01-01

    Accurate DNA segregation is essential for genome transmission. Segregation of the prototypical F plasmid requires the centromere-binding protein SopB, the NTPase SopA and the sopC centromere. SopB displays an intriguing range of DNA-binding properties essential for partition; it binds sopC to form a partition complex, which recruits SopA, and it also coats DNA to prevent non-specific SopA–DNA interactions, which inhibits SopA polymerization. To understand the myriad functions of SopB, we dete...

  17. Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

    Science.gov (United States)

    Sato, Wataru; Hitaoka, Seiji; Inoue, Kaoru; Imai, Mizue; Saio, Tomohide; Uchida, Takeshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yoshizawa, Kazunari; Ishimori, Koichiro

    2016-07-15

    Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.

  18. SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

    DEFF Research Database (Denmark)

    Lara, Enrique; Heger, Thierry J; Mitchell, Edward A D;

    2007-01-01

    The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study...... aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows...

  19. Pyrosequencing reveals the complex polymicrobial nature of invasive pyogenic infections: microbial constituents of empyema, liver abscess, and intracerebral abscess.

    Science.gov (United States)

    Sibley, C D; Church, D L; Surette, M G; Dowd, S E; Parkins, M D

    2012-10-01

    The polymicrobial nature of invasive pyogenic infections may be underestimated by routine culture practices, due to the fastidious nature of many organisms and the loss of viability during transport or from prior antibacterials. Pyrosequencing was performed on brain and liver abscesses and pleural fluid and compared to routine culture data. Forty-seven invasive pyogenic infection samples from 44 patients [6 intracerebral abscess (ICA), 21 pyogenic liver abscess (PLA), and 18 pleural fluid (PF) samples] were assayed. Pyrosequencing identified an etiologic microorganism in 100 % of samples versus 45 % by culture, p <0.01. Pyrosequencing was also more likely than traditional cultures to classify infections as polymicrobial, 91 % versus 17 %, p <0.001. The median number of genera identified by pyrosequencing compared to culture was 1 [interquartile range (IQR) 1-3] versus 0 (IQR 0-1) for ICA, 7 (IQR 1-15) versus 1 (IQR 0-1) for PLA, and 15 (IQR 9-19) versus 0 (IQR 0-1) for PF. Where organisms were cultured, they typically represented the numerically dominant species identified by pyrosequencing. Complex microbial communities are involved in invasive pyogenic infection of the lung, liver, and brain. Defining the polymicrobial nature of invasive pyogenic infections is the first step towards appreciating the clinical and diagnostic implications of these complex communities.

  20. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

    Directory of Open Access Journals (Sweden)

    David Llères

    2017-02-01

    Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

  1. Revealing and tuning the core, structure, properties and function of polymer micelles with lanthanide-coordination complexes.

    Science.gov (United States)

    Wang, Junyou; Groeneveld, Andrea; Oikonomou, Maria; Prusova, Alena; Van As, Henk; van Lent, Jan W M; Velders, Aldrik H

    2016-01-07

    Controlling self-assembly processes is of great interest in various fields where multifunctional and tunable materials are designed. We here present the versatility of lanthanide-complex-based micelles (Ln-C3Ms) with tunable coordination structures and corresponding functions (e.g. luminescence and magnetic relaxation enhancement). Micelles are prepared by charge-driven self-assembly of a polycationic-neutral diblock copolymer and anionic coordination complexes formed by Ln(III) ions and the bis-ligand L2EO4, which contains two dipicolinic acid (DPA) ligand groups (L) connected by a tetra-ethylene oxide spacer (EO4). By varying the DPA/Ln ratio, micelles are obtained with similar size but with different stability, different aggregation numbers and different oligomeric and polymeric lanthanide(III) coordination structures in the core. Electron microscopy, light scattering, luminescence spectroscopy and magnetic resonance relaxation experiments provide an unprecedented detailed insight into the core structures of such micelles. Concomitantly, the self-assembly is controlled such that tunable luminescence or magnetic relaxation with Eu-C3Ms, respectively, Gd-C3Ms is achieved, showing potential for applications, e.g. as contrast agents in (pre)clinical imaging. Considering the various lanthanide(III) ions have unique electron configurations with specific physical chemical properties, yet very similar coordination chemistry, the generality of the current coordination-structure based micellar design shows great promise for development of new materials such as, e.g., hypermodal agents.

  2. Complex network models reveal correlations among network metrics, exercise intensity and role of body changes in the fatigue process

    Science.gov (United States)

    Pereira, Vanessa Helena; Gama, Maria Carolina Traina; Sousa, Filipe Antônio Barros; Lewis, Theodore Gyle; Gobatto, Claudio Alexandre; Manchado-Gobatto, Fúlvia Barros

    2015-05-01

    The aims of the present study were analyze the fatigue process at distinct intensity efforts and to investigate its occurrence as interactions at distinct body changes during exercise, using complex network models. For this, participants were submitted to four different running intensities until exhaustion, accomplished in a non-motorized treadmill using a tethered system. The intensities were selected according to critical power model. Mechanical (force, peak power, mean power, velocity and work) and physiological related parameters (heart rate, blood lactate, time until peak blood lactate concentration (lactate time), lean mass, anaerobic and aerobic capacities) and IPAQ score were obtained during exercises and it was used to construction of four complex network models. Such models have both, theoretical and mathematical value, and enables us to perceive new insights that go beyond conventional analysis. From these, we ranked the influences of each node at the fatigue process. Our results shows that nodes, links and network metrics are sensibility according to increase of efforts intensities, been the velocity a key factor to exercise maintenance at models/intensities 1 and 2 (higher time efforts) and force and power at models 3 and 4, highlighting mechanical variables in the exhaustion occurrence and even training prescription applications.

  3. Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode.

    Science.gov (United States)

    Shimada, Satoru; Shinzawa-Itoh, Kyoko; Baba, Junpei; Aoe, Shimpei; Shimada, Atsuhiro; Yamashita, Eiki; Kang, Jiyoung; Tateno, Masaru; Yoshikawa, Shinya; Tsukihara, Tomitake

    2017-02-01

    Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c-CcO complex at 2.0-Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter-molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein-protein interaction at the docking interface represent the first known example of a new class of protein-protein interaction, which we term "soft and specific". This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c-CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

  4. Historical and contemporary geographic data reveal complex spatial and temporal responses of vegetation to climate and land stewardship

    Science.gov (United States)

    Villarreal, Miguel L.; Norman, Laura M.; Webb, Robert H.; Turner, Raymond M.

    2013-01-01

    Vegetation and land-cover changes are not always directional but follow complex trajectories over space and time, driven by changing anthropogenic and abiotic conditions. We present a multi-observational approach to land-change analysis that addresses the complex geographic and temporal variability of vegetation changes related to climate and land use. Using land-ownership data as a proxy for land-use practices, multitemporal land-cover maps, and repeat photography dating to the late 19th century, we examine changing spatial and temporal distributions of two vegetation types with high conservation value in the southwestern United States: grasslands and riparian vegetation. In contrast to many reported vegetation changes, notably shrub encroachment in desert grasslands, we found an overall increase in grassland area and decline of xeroriparian and riparian vegetation. These observed change patterns were neither temporally directional nor spatially uniform over the landscape. Historical data suggest that long-term vegetation changes coincide with broad climate fluctuations while fine-scale patterns are determined by land-management practices. In some cases, restoration and active management appear to weaken the effects of climate on vegetation; therefore, if land managers in this region act in accord with on-going directional changes, the current drought and associated ecological reorganization may provide an opportunity to achieve desired restoration endpoints.

  5. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

  6. The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments

    Institute of Scientific and Technical Information of China (English)

    Peng Wang; Di-Bing Sun; Bao-Li Qiu; Shu-Sheng Liu

    2011-01-01

    Recent phylogenetic analysis using mitochondrial cytochrome oxidase I(mtCOI)sequences of Bemisia tabaci worldwide indicates that the whitefly comprises at least 24 morphologically indistinguishable but genetically distinct cryptic species.While evidence of reproductive isolation has been reported for some of the putative species,more extensive crossing experiments are required to clarify the systematics of this species complex.In this study,we established laboratory cultures for six putative species of B.tabaci collected in China.We conducted 22 inter-species crosses among the six putative species.The data and those reported previously were collated,and the combined dataset covered all the 30 possible inter-species crosses among the six putative species.Intra-species controls always produced female and male progeny and the proportions of females in the first generation(F1)ranged from 56% to 70%.However,in inter-species crosses female progeny were rarely produced,and the few F1 females produced in four of the 30 inter-species crosses were either sterile or significantly weaker in viability.These results demonstrate a pattern of complete reproductive isolation among the six putative species and show that they are six cryptic species in the B.tabaci complex.

  7. Complex network models reveal correlations among network metrics, exercise intensity and role of body changes in the fatigue process.

    Science.gov (United States)

    Pereira, Vanessa Helena; Gama, Maria Carolina Traina; Sousa, Filipe Antônio Barros; Lewis, Theodore Gyle; Gobatto, Claudio Alexandre; Manchado-Gobatto, Fúlvia Barros

    2015-05-21

    The aims of the present study were analyze the fatigue process at distinct intensity efforts and to investigate its occurrence as interactions at distinct body changes during exercise, using complex network models. For this, participants were submitted to four different running intensities until exhaustion, accomplished in a non-motorized treadmill using a tethered system. The intensities were selected according to critical power model. Mechanical (force, peak power, mean power, velocity and work) and physiological related parameters (heart rate, blood lactate, time until peak blood lactate concentration (lactate time), lean mass, anaerobic and aerobic capacities) and IPAQ score were obtained during exercises and it was used to construction of four complex network models. Such models have both, theoretical and mathematical value, and enables us to perceive new insights that go beyond conventional analysis. From these, we ranked the influences of each node at the fatigue process. Our results shows that nodes, links and network metrics are sensibility according to increase of efforts intensities, been the velocity a key factor to exercise maintenance at models/intensities 1 and 2 (higher time efforts) and force and power at models 3 and 4, highlighting mechanical variables in the exhaustion occurrence and even training prescription applications.

  8. Multiscale models of skeletal muscle reveal the complex effects of muscular dystrophy on tissue mechanics and damage susceptibility.

    Science.gov (United States)

    Virgilio, Kelley M; Martin, Kyle S; Peirce, Shayn M; Blemker, Silvia S

    2015-04-06

    Computational models have been increasingly used to study the tissue-level constitutive properties of muscle microstructure; however, these models were not created to study or incorporate the influence of disease-associated modifications in muscle. The purpose of this paper was to develop a novel multiscale muscle modelling framework to elucidate the relationship between microstructural disease adaptations and modifications in both mechanical properties of muscle and strain in the cell membrane. We used an agent-based model to randomly generate new muscle fibre geometries and mapped them into a finite-element model representing a cross section of a muscle fascicle. The framework enabled us to explore variability in the shape and arrangement of fibres, as well as to incorporate disease-related changes. We applied this method to reveal the trade-offs between mechanical properties and damage susceptibility in Duchenne muscular dystrophy (DMD). DMD is a fatal genetic disease caused by a lack of the transmembrane protein dystrophin, leading to muscle wasting and death due to cardiac or pulmonary complications. The most prevalent microstructural variations in DMD include: lack of transmembrane proteins, fibrosis, fatty infiltration and variation in fibre cross-sectional area. A parameter analysis of these variations and case study of DMD revealed that the nature of fibrosis and density of transmembrane proteins strongly affected the stiffness of the muscle and susceptibility to membrane damage.

  9. Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

    Science.gov (United States)

    D'Ovidio, Renato; Raiola, Alessandro; Capodicasa, Cristina; Devoto, Alessandra; Pontiggia, Daniela; Roberti, Serena; Galletti, Roberta; Conti, Eric; O'Sullivan, Donal; De Lorenzo, Giulia

    2004-08-01

    Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

  10. Joint cross-correlation analysis reveals complex, time-dependent functional relationship between cortical neurons and arm electromyograms

    Science.gov (United States)

    Zhuang, Katie Z.; Lebedev, Mikhail A.

    2014-01-01

    Correlation between cortical activity and electromyographic (EMG) activity of limb muscles has long been a subject of neurophysiological studies, especially in terms of corticospinal connectivity. Interest in this issue has recently increased due to the development of brain-machine interfaces with output signals that mimic muscle force. For this study, three monkeys were implanted with multielectrode arrays in multiple cortical areas. One monkey performed self-timed touch pad presses, whereas the other two executed arm reaching movements. We analyzed the dynamic relationship between cortical neuronal activity and arm EMGs using a joint cross-correlation (JCC) analysis that evaluated trial-by-trial correlation as a function of time intervals within a trial. JCCs revealed transient correlations between the EMGs of multiple muscles and neural activity in motor, premotor and somatosensory cortical areas. Matching results were obtained using spike-triggered averages corrected by subtracting trial-shuffled data. Compared with spike-triggered averages, JCCs more readily revealed dynamic changes in cortico-EMG correlations. JCCs showed that correlation peaks often sharpened around movement times and broadened during delay intervals. Furthermore, JCC patterns were directionally selective for the arm-reaching task. We propose that such highly dynamic, task-dependent and distributed relationships between cortical activity and EMGs should be taken into consideration for future brain-machine interfaces that generate EMG-like signals. PMID:25210153

  11. 16S rRNA survey revealed complex bacterial communities and evidence of bacterial interference on human adenoids.

    Science.gov (United States)

    Ren, Tiantian; Glatt, Dominique Ulrike; Nguyen, Tam Nhu; Allen, Emma Kaitlynn; Early, Stephen V; Sale, Michele; Winther, Birgit; Wu, Martin

    2013-02-01

    Adenoid microbiota plays an important role in the development of various infectious and non-infectious diseases of the upper airways, such as otitis media, adenotonsillitis, rhinosinusitis and adenoid hypertrophy. Studies have suggested that adenoids could act as a potential reservoir of opportunistic pathogens. However, previous bacterial surveys of adenoids were mainly culture based and therefore might only provide an incomplete and potentially biased assessment of the microbial diversity. To develop an in-depth and comprehensive understanding of the adenoid microbial communities and test the 'pathogen reservoir hypothesis', we carried out a 16S rRNA based, culture-independent survey of bacterial communities on 67 human adenoids removed by surgery. Our survey revealed highly diverse adenoid bacterial communities distinct from those of other body habitats. Despite large interpersonal variations, adenoid microbiota shared a core set of taxa and can be classified into at least five major types based on its bacterial species composition. Our results support the 'pathogen reservoir hypothesis' as we found common pathogens of otitis media to be both prevalent and abundant. Co-occurrence analyses revealed evidence consistent with the bacterial interference theory in that multiple common pathogens showed 'non-coexistence' relationships with non-pathogenic members of the commensal microflora.

  12. Systematics and biogeography of the Automolus infuscatus complex (Aves; Furnariidae): Cryptic diversity reveals western Amazonia as the origin of a transcontinental radiation.

    Science.gov (United States)

    Schultz, Eduardo D; Burney, Curtis W; Brumfield, Robb T; Polo, Erico M; Cracraft, Joel; Ribas, Camila C

    2017-02-01

    A revision of the avian Neotropical genus Automolus and the Furnariidae family points to the paraphyly of A. infuscatus and reveals a species complex comprising A. infuscatus, A. ochrolaemus, A. paraensis, A. leucophthalmus, A. lammi and A. subulatus, the latter historically classified in the genus Hyloctistes. Detailed knowledge of the taxonomy, geographic distribution, phylogenetic relationship and divergence times of a taxon allows exploration of its evolutionary history and the testing of different scenarios of diversification. In this context, we studied the A. infuscatus complex using molecular data in order to unveil its cryptic diversity and reveal its evolutionary history. For that we sequenced two mitochondrial (ND2 and cytb) and three nuclear markers (G3PDH, ACO, Fib7) for 302 individuals belonging to all species in the complex and most described subspecies. Our analysis supports the paraphyly of A. infuscatus, indicating the existence of at least two distinct clades not closely related. The remaining species were all recovered as monophyletic. Notwithstanding, a well-structured intraspecific diversity was found with 19 lineages suggesting substantial cryptic diversity within the described species. A. subulatus was recovered within the complex, corroborating its position inside the genus. In spite of the high congruence between distributions of different lineages, with several sister lineages currently separated by the same barriers, the temporal incongruence between divergences over the same barriers reveals a complex evolutionary history. While older events might be related to the emergence of barriers such as the Andes and major Amazonian rivers, younger events suggest dispersal after the consolidation of those barriers. Our analysis suggests that the complex had its origin around 6million years (Ma) and inhabited Western Amazonia in Late Miocene-Early Pliocene. Considering the riparian habit of species in its sister clade, the rise and early

  13. Integrative taxonomy of the Russet Bush Warbler Locustella mandelli complex reveals a new species from central China

    Institute of Scientific and Technical Information of China (English)

    Per; Alstr?m; Canwei; Xia; Pamela; C; Rasmussen; Urban; Olsson; Bo; Dai; Jian; Zhao; Paul; J; Leader; Geoff; J; Carey; Lu; Dong; Tianlong; Cai; Paul; I; Holt; Hung; Le; Manh; Gang; Song; Yang; Liu; Yanyun; Zhang; Fumin; Lei

    2015-01-01

    Background: The Russet Bush Warbler Locustella(previously Bradypterus) mandelli complex occurs in mountains in the eastern Himalayas, southern China, Vietnam, the Philippines, and Indonesia. The taxonomy has been debated,with one(L. seebohmi) to four(L. seebohmi, L. mandelli, L. montis and L. timorensis) species having been recognised.Methods: We used an integrative approach, incorporating analyses of morphology, vocalizations and a molecular marker, to re-evaluate species limits in the L. mandelli complex.Results: We found that central Chinese L. mandelli differed from those from India through northern Southeast Asia to southeast China in plumage, morphometrics and song. All were easily classified by song, and(wing + culmen)/tail ratio overlapped only marginally. Both groups were reciprocally monophyletic in a mitochondrial cytochrome b(cytb) gene tree, with a mean divergence of 1.0 ± 0.2%. They were sympatric and mostly altitudinally segregated in the breeding season in southern Sichuan province. We found that the Mt Victoria(western Myanmar) population differed vocally from other L. mandelli, but no specimens are available. Taiwan Bush Warbler L. alishanensis was sister to the L. mandelli complex, with the most divergent song. Plumage, vocal and cytb evidence supported the distinctness of the south Vietnamese L. mandelli idonea. The Timor Bush Warbler L. timorensis, Javan Bush Warbler L.montis and Benguet Bush Warbler L. seebohmi differed distinctly in plumage, but among-population song variation in L. montis exceeded the differences between some populations of these taxa, and mean pairwise cytb divergences were only 0.5–0.9%. We also found that some L. montis populations differed morphologically.Conclusions: We conclude that the central Chinese population of Russet Bush Warbler represents a new species,which we describe herein, breeding at mid elevations in Sichuan, Shaanxi, Hubei, Hunan and Guizhou provinces.The taxonomic status of the other allopatric

  14. Integrative taxonomy of the Russet Bush Warbler Locustella mandelli complex reveals a new species from central China

    Institute of Scientific and Technical Information of China (English)

    Per Alström; Tianlong Cai1; Paul I Holt; Hung Le Manh; Gang Song; Yang Liu; Yanyun Zhang; Fumin Lei; Canwei Xia; Pamela C Rasmussen; Urban Olsson; Bo Dai; Jian Zhao; Paul J Leader; Geoff J Carey; Lu Dong

    2015-01-01

    Background:The Russet Bush Warbler Locustel a (previously Bradypterus) mandel i complex occurs in mountains in the eastern Himalayas, southern China, Vietnam, the Philippines, and Indonesia. The taxonomy has been debated, with one (L. seebohmi) to four (L. seebohmi, L. mandel i, L. montis and L. timorensis) species having been recognised. Methods:We used an integrative approach, incorporating analyses of morphology, vocalizations and a molecular marker, to re-evaluate species limits in the L. mandel i complex. Results:We found that central Chinese L. mandel i differed from those from India through northern Southeast Asia to southeast China in plumage, morphometrics and song. All were easily classified by song, and (wing+culmen)/tail ratio overlapped only marginally. Both groups were reciprocally monophyletic in a mitochondrial cytochrome b (cytb) gene tree, with a mean divergence of 1.0 ± 0.2%. They were sympatric and mostly altitudinally segregated in the breeding season in southern Sichuan province. We found that the Mt Victoria (western Myanmar) population differed vocally from other L. mandel i, but no specimens are available. Taiwan Bush Warbler L. alishanensis was sister to the L. mandel i complex, with the most divergent song. Plumage, vocal and cytb evidence supported the distinctness of the south Vietnamese L. mandel i idonea. The Timor Bush Warbler L. timorensis, Javan Bush Warbler L. montis and Benguet Bush Warbler L. seebohmi differed distinctly in plumage, but among-population song variation in L. montis exceeded the differences between some populations of these taxa, and mean pairwise cytb divergences were only 0.5–0.9%. We also found that some L. montis populations differed morphologically. Conclusions:We conclude that the central Chinese population of Russet Bush Warbler represents a new species, which we describe herein, breeding at mid elevations in Sichuan, Shaanxi, Hubei, Hunan and Guizhou provinces. The taxonomic status of the other

  15. Transitions in social complexity along elevational gradients reveal a combined impact of season length and development time on social evolution.

    Science.gov (United States)

    Kocher, Sarah D; Pellissier, Loïc; Veller, Carl; Purcell, Jessica; Nowak, Martin A; Chapuisat, Michel; Pierce, Naomi E

    2014-07-22

    Eusociality is taxonomically rare, yet associated with great ecological success. Surprisingly, studies of environmental conditions favouring eusociality are often contradictory. Harsh conditions associated with increasing altitude and latitude seem to favour increased sociality in bumblebees and ants, but the reverse pattern is found in halictid bees and polistine wasps. Here, we compare the life histories and distributions of populations of 176 species of Hymenoptera from the Swiss Alps. We show that differences in altitudinal distributions and development times among social forms can explain these contrasting patterns: highly social taxa develop more quickly than intermediate social taxa, and are thus able to complete the reproductive cycle in shorter seasons at higher elevations. This dual impact of altitude and development time on sociality illustrates that ecological constraints can elicit dynamic shifts in behaviour, and helps explain the complex distribution of sociality across ecological gradients.

  16. Crystal structures of PRK1 in complex with the clinical compounds lestaurtinib and tofacitinib reveal ligand induced conformational changes.

    Science.gov (United States)

    Chamberlain, Philip; Delker, Silvia; Pagarigan, Barbra; Mahmoudi, Afshin; Jackson, Pilgrim; Abbasian, Mahan; Muir, Jeff; Raheja, Neil; Cathers, Brian

    2014-01-01

    Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

  17. Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci.

    Science.gov (United States)

    Martin, Paul; McGovern, Amanda; Orozco, Gisela; Duffus, Kate; Yarwood, Annie; Schoenfelder, Stefan; Cooper, Nicholas J; Barton, Anne; Wallace, Chris; Fraser, Peter; Worthington, Jane; Eyre, Steve

    2015-11-30

    Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions and provide evidence that only a minority of interactions are common to both B- and T-cell lines, suggesting interactions may be highly cell-type specific; some disease-associated SNPs do not interact with the nearest gene but with more compelling candidate genes (for example, FOXO1, AZI2) often situated several megabases away; and finally, regions associated with different autoimmune diseases interact with each other and the same promoter suggesting common autoimmune gene targets (for example, PTPRC, DEXI and ZFP36L1).

  18. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.

    Science.gov (United States)

    Mouilleron, Stéphane; Langer, Carola A; Guettler, Sebastian; McDonald, Neil Q; Treisman, Richard

    2011-06-14

    Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.

  19. Genome-wide macrosynteny among Fusarium species in the Gibberella fujikuroi complex revealed by amplified fragment length polymorphisms.

    Science.gov (United States)

    De Vos, Lieschen; Steenkamp, Emma T; Martin, Simon H; Santana, Quentin C; Fourie, Gerda; van der Merwe, Nicolaas A; Wingfield, Michael J; Wingfield, Brenda D

    2014-01-01

    The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

  20. Multistructure index in revealing complexity of regulatory mechanisms of human cardiovascular system at rest and orthostatic stress in healthy humans

    Science.gov (United States)

    Makowiec, Danuta; Graff, Beata; Struzik, Zbigniew R.

    2017-02-01

    Biological regulation is sufficiently complex to pose an enduring challenge for characterization of both its equilibrium and transient non-equilibrium dynamics. Two univariate but coupled observables, heart rate and systolic blood pressure, are commonly characterized in the benchmark example of the human cardiovascular regulatory system. Asymmetric distributions of accelerations and decelerations of heart rate, as well as rises and falls in systolic blood pressure, recorded in humans during a head-up tilt test provide insights into the dynamics of cardiovascular response to a rapid, controlled deregulation of the system's homeostasis. The baroreflex feedback loop is assumed to be the fundamental physiological mechanism for ensuring homeostatic blood supply to distant organs at rest and during orthostatic stress, captured in a classical beat-to-beat autoregressive model of baroreflex by de Boer et al. (1987). For model corroboration, a multistructure index statistic is proposed, seamlessly evaluating the size spectrum of magnitudes of neural reflexes such as baroreflex, responsible for maintaining the homeostatic dynamics. The multistructure index exposes a distinctly different dynamics of multiscale asymmetry between results obtained from real-life signals recorded from healthy subjects and those simulated using both the classical and perturbed versions of the model. Nonlinear effects observed suggest the pronounced presence of complex mechanisms resulting from baroreflex regulation when a human is at rest, which is aggravated in the system's response to orthostatic stress. Using our methodology of multistructure index, we therefore show a marked difference between model and real-life scenarios, which we attribute to multiscale asymmetry of non-linear origin in real-life signals, which we are not reproducible by the classical model.

  1. A Polychaete’s Powerful Punch: Venom Gland Transcriptomics of Glycera Reveals a Complex Cocktail of Toxin Homologs

    Science.gov (United States)

    von Reumont, Björn M.; Richter, Sandy; Hering, Lars; Sykes, Dan; Hetmank, Jörg; Jenner, Ronald A.; Bleidorn, Christoph

    2014-01-01

    Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands. PMID:25193302

  2. Dynamics of metal-humate complexation equilibria as revealed by isotope exchange studies - a matter of concentration and time

    Science.gov (United States)

    Lippold, Holger; Eidner, Sascha; Kumke, Michael U.; Lippmann-Pipke, Johanna

    2017-01-01

    Complexation with dissolved humic matter can be crucial in controlling the mobility of toxic or radioactive contaminant metals. For speciation and transport modelling, a dynamic equilibrium process is commonly assumed, where association and dissociation run permanently. This is, however, questionable in view of reported observations of a growing resistance to dissociation over time. In this study, the isotope exchange principle was employed to gain direct insight into the dynamics of the complexation equilibrium, including kinetic inertisation phenomena. Terbium(III), an analogue of trivalent actinides, was used as a representative of higher-valent metals. Isotherms of binding to (flocculated) humic acid, determined by means of 160Tb as a radiotracer, were found to be identical regardless of whether the radioisotope was introduced together with the bulk of stable 159Tb or subsequently after pre-equilibration for up to 3 months. Consequently, there is a permanent exchange of free and humic-bound Tb since all available binding sites are occupied in the plateau region of the isotherm. The existence of a dynamic equilibrium was thus evidenced. There was no indication of an inertisation under these experimental conditions. If the small amount of 160Tb was introduced prior to saturation with 159Tb, the expected partial desorption of 160Tb occurred at much lower rates than observed for the equilibration process in the reverse procedure. In addition, the rates decreased with time of pre-equilibration. Inertisation phenomena are thus confined to the stronger sites of humic molecules (occupied at low metal concentrations). Analysing the time-dependent course of isotope exchange according to first-order kinetics indicated that up to 3 years are needed to attain equilibrium. Since, however, metal-humic interaction remains reversible, exchange of metals between humic carriers and mineral surfaces cannot be neglected on the long time scale to be considered in predictive

  3. Long non-coding RNA discovery across the genus anopheles reveals conserved secondary structures within and beyond the Gambiae complex.

    Science.gov (United States)

    Jenkins, Adam M; Waterhouse, Robert M; Muskavitch, Marc A T

    2015-04-23

    Long non-coding RNAs (lncRNAs) have been defined as mRNA-like transcripts longer than 200 nucleotides that lack significant protein-coding potential, and many of them constitute scaffolds for ribonucleoprotein complexes with critical roles in epigenetic regulation. Various lncRNAs have been implicated in the modulation of chromatin structure, transcriptional and post-transcriptional gene regulation, and regulation of genomic stability in mammals, Caenorhabditis elegans, and Drosophila melanogaster. The purpose of this study is to identify the lncRNA landscape in the malaria vector An. gambiae and assess the evolutionary conservation of lncRNAs and their secondary structures across the Anopheles genus. Using deep RNA sequencing of multiple Anopheles gambiae life stages, we have identified 2,949 lncRNAs and more than 300 previously unannotated putative protein-coding genes. The lncRNAs exhibit differential expression profiles across life stages and adult genders. We find that across the genus Anopheles, lncRNAs display much lower sequence conservation than protein-coding genes. Additionally, we find that lncRNA secondary structure is highly conserved within the Gambiae complex, but diverges rapidly across the rest of the genus Anopheles. This study offers one of the first lncRNA secondary structure analyses in vector insects. Our description of lncRNAs in An. gambiae offers the most comprehensive genome-wide insights to date into lncRNAs in this vector mosquito, and defines a set of potential targets for the development of vector-based interventions that may further curb the human malaria burden in disease-endemic countries.

  4. Complex network analysis of CA3 transcriptome reveals pathogenic and compensatory pathways in refractory temporal lobe epilepsy.

    Directory of Open Access Journals (Sweden)

    Silvia Yumi Bando

    Full Text Available We previously described - studying transcriptional signatures of hippocampal CA3 explants - that febrile (FS and afebrile (NFS forms of refractory mesial temporal lobe epilepsy constitute two distinct genomic phenotypes. That network analysis was based on a limited number (hundreds of differentially expressed genes (DE networks among a large set of valid transcripts (close to two tens of thousands. Here we developed a methodology for complex network visualization (3D and analysis that allows the categorization of network nodes according to distinct hierarchical levels of gene-gene connections (node degree and of interconnection between node neighbors (concentric node degree. Hubs are highly connected nodes, VIPs have low node degree but connect only with hubs, and high-hubs have VIP status and high overall number of connections. Studying the whole set of CA3 valid transcripts we: i obtained complete transcriptional networks (CO for FS and NFS phenotypic groups; ii examined how CO and DE networks are related; iii characterized genomic and molecular mechanisms underlying FS and NFS phenotypes, identifying potential novel targets for therapeutic interventions. We found that: i DE hubs and VIPs are evenly distributed inside the CO networks; ii most DE hubs and VIPs are related to synaptic transmission and neuronal excitability whereas most CO hubs, VIPs and high hubs are related to neuronal differentiation, homeostasis and neuroprotection, indicating compensatory mechanisms. Complex network visualization and analysis is a useful tool for systems biology approaches to multifactorial diseases. Network centrality observed for hubs, VIPs and high hubs of CO networks, is consistent with the network disease model, where a group of nodes whose perturbation leads to a disease phenotype occupies a central position in the network. Conceivably, the chance for exerting therapeutic effects through the modulation of particular genes will be higher if these genes

  5. Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

    Directory of Open Access Journals (Sweden)

    Derry Voisin

    2009-10-01

    Full Text Available Mutations in LACERATA (LCR, FIDDLEHEAD (FDH, and BODYGUARD (BDG cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis, of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE, which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

  6. General interaction mode of CIDE:CIDE complex revealed by a mutation study of the Drep2 CIDE domain.

    Science.gov (United States)

    Lee, Seung Mi; Park, Hyun Ho

    2013-04-02

    The CIDE domain is a well known protein-protein interaction module that is initially detected at the apoptotic DNA fragmentation factor (DFF40/45). The interaction mechanism via the CIDE domain is not well understood. To elucidate CIDE domain mediated interactions in the apoptotic DNA fragmentation system, we conducted biochemical and mutational studies and found that the surface of CIDE domains can be divided into an acidic side and a basic side. In addition, a mutagenesis study revealed that the basic surface side of Drep2 CIDE is involved in the interaction with the acidic surface side of Drep1 CIDE and Drep3 CIDE. Our research supports the idea that a charge-charge interaction might be the general interaction mode of the CIDE:CIDE interaction.

  7. Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus*♦

    Science.gov (United States)

    Dickson, Claire F.; Kumar, Kaavya Krishna; Jacques, David A.; Malmirchegini, G. Reza; Spirig, Thomas; Mackay, Joel P.; Clubb, Robert T.; Guss, J. Mitchell; Gell, David A.

    2014-01-01

    Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins. PMID:24425866

  8. Structure of the hemoglobin-IsdH complex reveals the molecular basis of iron capture by Staphylococcus aureus.

    Science.gov (United States)

    Dickson, Claire F; Kumar, Kaavya Krishna; Jacques, David A; Malmirchegini, G Reza; Spirig, Thomas; Mackay, Joel P; Clubb, Robert T; Guss, J Mitchell; Gell, David A

    2014-03-01

    Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.

  9. Characterization of the effects of Escherichia coli replication terminator protein (Tus) on transcription reveals dynamic nature of the tus block to transcription complex progression.

    Science.gov (United States)

    Guajardo, R; Sousa, R

    1999-01-01

    We have characterized the blocks to progression of T7 and T3 RNA polymerase transcription complexes created when a Tus protein is bound to the template. The encounter with Tus impedes the progress of the transcription complexes of either enzyme. The duration of the block depends on which polymerase is used and the orientation of Tus on the DNA. Both genuine termination (dissociation of the transcription complex) and halting followed by continued progression after the block is abrogated are observed. The fraction of complexes that terminates depends on which polymerase is used and on the orientation of the Tus molecule. The efficiency of the block to transcription increases as the Tus concentration is increased, even if the concentration of Tus is already many times in excess of what is required to saturate its binding sites on the template in the absence of transcription. The block to transcription is rapidly abrogated if an excess of a DNA containing a binding site for Tus is added to a transcription reaction in which Tus and template have been preincubated. Finally, we find that transcription will rapidly displace Tus from a template under conditions that generate persistent blocks to transcription. These observations reveal that during the encounter with the transcription complex Tus rapidly dissociates from the template but that at sufficiently high concentrations Tus usually rebinds before the transcription complex can move forward. The advantage of a mechanism which can create a persistent block to transcription or replication complex progression, which can nevertheless be rapidly abrogated in response to down regulation of the blocking protein, is suggested. PMID:10373601

  10. Structure of UreG/UreF/UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter pylori urease.

    Directory of Open Access Journals (Sweden)

    Yu Hang Fong

    2013-10-01

    Full Text Available Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.

  11. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  12. Crystal Structure of the VP4 Protease from Infectious Pancreatic Necrosis Virus Reveals the acyl-enzyme Complex for an Intermolecular Self-Cleavage Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Lee,J.; Feldman, A.; Delmas, B.; Paetzel, M.

    2007-01-01

    Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH{sub 2}-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-{angstrom} resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser{sup 633}O{gamma} forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala{sup 716}, of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-{angstrom} resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ({approx}19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.

  13. Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis)

    Science.gov (United States)

    Sun, Ping; Cheng, Chunzhen; Lin, Yuling; Zhu, Qiufang; Lin, Jinke

    2017-01-01

    MicroRNAs are endogenous non-coding small RNAs playing crucial regulatory roles in plants. Tea, a globally popular non-alcoholic drink, is rich in health-enhancing catechins. In this study, 69 conserved and 47 novel miRNAs targeting 644 genes were identified by high-throughout sequencing. Predicted target genes of miRNAs were mainly involved in plant growth, signal transduction, morphogenesis and defense. To further identify targets of tea miRNAs, degradome sequencing and RNA ligase-mediated rapid amplification of 5’cDNA ends (RLM-RACE) were applied. Using degradome sequencing, 26 genes mainly involved in transcription factor, resistance protein and signal transduction protein synthesis were identified as potential miRNA targets, with 5 genes subsequently verified. Quantitative real-time PCR (qRT-PCR) revealed that the expression patterns of novel-miR1, novel-miR2, csn-miR160a, csn-miR162a, csn-miR394 and csn-miR396a were negatively correlated with catechin content. The expression of six miRNAs (csn-miRNA167a, csn-miR2593e, csn-miR4380a, csn-miR3444b, csn-miR5251 and csn-miR7777-5p.1) and their target genes involved in catechin biosynthesis were also analyzed by qRT-PCR. Negative and positive correlations were found between these miRNAs and catechin contents, while positive correlations were found between their target genes and catechin content. This result suggests that these miRNAs may negatively regulate catechin biosynthesis by down-regulating their biosynthesis-related target genes. Taken together, our results indicate that miRNAs are crucial regulators in tea, with the results of 5’-RLM-RACE and expression analyses revealing the important role of miRNAs in catechin anabolism. Our findings should facilitate future research to elucidate the function of miRNAs in catechin biosynthesis. PMID:28225779

  14. Structure of a Clostridium botulinum C143S thiaminase I/thiamin complex reveals active site architecture .

    Science.gov (United States)

    Sikowitz, Megan D; Shome, Brateen; Zhang, Yang; Begley, Tadhg P; Ealick, Steven E

    2013-11-05

    Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II.

  15. The complex nature of superconductivity in MgB2 as revealed by the reduced total isotope effect.

    Science.gov (United States)

    Hinks, D G; Claus, H; Jorgensen, J D

    2001-05-24

    Magnesium diboride, MgB2, was recently observed to become superconducting at 39 K, which is the highest known transition temperature for a non-copper-oxide bulk material. Isotope-effect measurements, in which atoms are substituted by isotopes of different mass to systematically change the phonon frequencies, are one of the fundamental tests of the nature of the superconducting mechanism in a material. In a conventional Bardeen-Cooper-Schrieffer (BCS) superconductor, where the mechanism is mediated by electron-phonon coupling, the total isotope-effect coefficient (in this case, the sum of both the Mg and B coefficients) should be about 0.5. The boron isotope effect was previously shown to be large and that was sufficient to establish that MgB2 is a conventional superconductor, but the Mg effect has not hitherto been measured. Here we report the determination of the Mg isotope effect, which is small but measurable. The total reduced isotope-effect coefficient is 0.32, which is much lower than the value expected for a typical BCS superconductor. The low value could be due to complex materials properties, and would seem to require both a large electron-phonon coupling constant and a value of mu* (the repulsive electron-electron interaction) larger than found for most simple metals.

  16. The probabilistic niche model reveals the niche structure and role of body size in a complex food web.

    Directory of Open Access Journals (Sweden)

    Richard J Williams

    Full Text Available The niche model has been widely used to model the structure of complex food webs, and yet the ecological meaning of the single niche dimension has not been explored. In the niche model, each species has three traits, niche position, diet position and feeding range. Here, a new probabilistic niche model, which allows the maximum likelihood set of trait values to be estimated for each species, is applied to the food web of the Benguela fishery. We also developed the allometric niche model, in which body size is used as the niche dimension. About 80% of the links in the empirical data are predicted by the probabilistic niche model, a significant improvement over recent models. As in the niche model, species are uniformly distributed on the niche axis. Feeding ranges are exponentially distributed, but diet positions are not uniformly distributed below the predator. Species traits are strongly correlated with body size, but the allometric niche model performs significantly worse than the probabilistic niche model. The best-fit parameter set provides a significantly better model of the structure of the Benguela food web than was previously available. The methodology allows the identification of a number of taxa that stand out as outliers either in the model's poor performance at predicting their predators or prey or in their parameter values. While important, body size alone does not explain the structure of the one-dimensional niche.

  17. A novel 3-D bio-microfluidic system mimicking in vivo heterogeneous tumour microstructures reveals complex tumour–stroma interactions

    KAUST Repository

    Fan, Qihui

    2017-07-10

    A 3-D microfluidic system consisting of microchamber arrays embedded in a collagen hydrogel with tuneable biochemical gradients that mimics the tumour microenvironment of mammary glands was constructed for the investigation on the interactions between invasive breast cancer cells and stromal cells. The hollow microchambers in collagen provide a very similar 3-D environment to that in vivo that regulates collective cellular dynamics and behaviour, while the microfluidic channels surrounding the collagen microchamber arrays allow one to impose complex concentration gradients of specific biological molecules or drugs. We found that breast epithelial cells (MCF-10A) seeded in the microchambers formed lumen-like structures similar to those in epithelial layers. When MCF-10A cells were co-cultured with invasive breast cancer cells (MDA-MB-231), the formation of lumen-like structures in the microchambers was inhibited, indicating the capability of cancer cells to disrupt the structures formed by surrounding cells for further invasion and metastasis. Subsequent mechanism studies showed that down regulation of E-cad expression due to MMPs produced by the cancer cells plays a dominant role in determining the cellular behaviour. Our microfluidic system offers a robust platform for high throughput studies that aim to understand combinatorial effects of multiple biochemical and microenvironmental factors.

  18. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  19. Phylogenies of central element proteins reveal the dynamic evolutionary history of the mammalian synaptonemal complex: ancient and recent components.

    Science.gov (United States)

    Fraune, Johanna; Brochier-Armanet, Céline; Alsheimer, Manfred; Benavente, Ricardo

    2013-11-01

    During meiosis, the stable pairing of the homologous chromosomes is mediated by the assembly of the synaptonemal complex (SC). Its tripartite structure is well conserved in Metazoa and consists of two lateral elements (LEs) and a central region (CR) that in turn is formed by several transverse filaments (TFs) and a central element (CE). In a previous article, we have shown that not only the structure, but also the major structural proteins SYCP1 (TFs) and SYCP3 (LEs) of the mammalian SC are conserved in metazoan evolution. In continuation of this work, we now investigated the evolution of the mammalian CE-specific proteins using phylogenetic and biochemical/cytological approaches. In analogy to the observations made for SYCP1 and SYCP3, we did not detect homologs of the mammalian CE proteins in insects or nematodes, but in several other metazoan clades. We were able to identify homologs of three mammalian CE proteins in several vertebrate and invertebrate species, for two of these proteins down to the basal-branching phylum of Cnidaria. Our approaches indicate that the SC arose only once, but evolved dynamically during diversification of Metazoa. Certain proteins appear to be ancient in animals, but successive addition of further components as well as protein loss and/or replacements have also taken place in some lineages.

  20. Genomewide effects of peroxisome proliferator-activated receptor gamma in macrophages and dendritic cells--revealing complexity through systems biology.

    Science.gov (United States)

    Cuaranta-Monroy, Ixchelt; Kiss, Mate; Simandi, Zoltan; Nagy, Laszlo

    2015-09-01

    Systems biology approaches have become indispensable tools in biomedical and basic research. These data integrating bioinformatic methods gained prominence after high-throughput technologies became available to investigate complex cellular processes, such as transcriptional regulation and protein-protein interactions, on a scale that had not been studied before. Immunology is one of the medical fields that systems biology impacted profoundly due to the plasticity of cell types involved and the accessibility of a wide range of experimental models. In this review, we summarize the most important recent genomewide studies exploring the function of peroxisome proliferator-activated receptor γ in macrophages and dendritic cells. PPARγ ChIP-seq experiments were performed in adipocytes derived from embryonic stem cells to complement the existing data sets and to provide comparators to macrophage data. Finally, lists of regulated genes generated from such experiments were analysed with bioinformatics and system biology approaches. We show that genomewide studies utilizing high-throughput data acquisition methods made it possible to gain deeper insights into the role of PPARγ in these immune cell types. We also demonstrate that analysis and visualization of data using network-based approaches can be used to identify novel genes and functions regulated by the receptor. The example of PPARγ in macrophages and dendritic cells highlights the crucial importance of systems biology approaches in establishing novel cellular functions for long-known signaling pathways. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

  1. Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex

    Science.gov (United States)

    Li, Peng; Wang, Dechen; Yan, Jinli; Zhou, Jianuan; Deng, Yinyue; Jiang, Zide; Cao, Bihao; He, Zifu; Zhang, Lianhui

    2016-01-01

    Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species. PMID:27833603

  2. Molecular epidemiology of infectious hematopoietic necrosis virus reveals complex virus traffic and evolution within southern Idaho aquaculture

    Science.gov (United States)

    Troyer, R.M.; Kurath, G.

    2003-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which infects salmon and trout and may cause disease with up to 90% mortality. In the Hagerman Valley of Idaho, IHNV is endemic or epidemic among numerous fish farms and resource mitigation hatcheries. A previous study characterizing the genetic diversity among 84 IHNV isolates at 4 virus-endemic rainbow trout farms indicated that multiple lineages of relatively high diversity co-circulated at these facilities (Troyer et al. 2000 J Gen Virol. 81:2823-2832). We tested the hypothesis that high IHNV genetic diversity and co-circulating lineages are present in aquaculture facilities throughout this region. In this study, 73 virus isolates from 14 rainbow trout farms and 3 state hatcheries in the Hagerman Valley, isolated between 1978 and 1999, were genetically characterized by sequence analysis of a 303 nucleotide region of the glycoprotein gene. Phylogenetic and epidemiological analyses showed that multiple IHNV lineages co-circulate in a complex pattern throughout private trout farms and state hatcheries in the valley. IHNV maintained within the valley appears to have evolved significantly over the 22 yr study period.

  3. Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci

    Science.gov (United States)

    Martin, Paul; McGovern, Amanda; Orozco, Gisela; Duffus, Kate; Yarwood, Annie; Schoenfelder, Stefan; Cooper, Nicholas J.; Barton, Anne; Wallace, Chris; Fraser, Peter; Worthington, Jane; Eyre, Steve

    2015-01-01

    Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions and provide evidence that only a minority of interactions are common to both B- and T-cell lines, suggesting interactions may be highly cell-type specific; some disease-associated SNPs do not interact with the nearest gene but with more compelling candidate genes (for example, FOXO1, AZI2) often situated several megabases away; and finally, regions associated with different autoimmune diseases interact with each other and the same promoter suggesting common autoimmune gene targets (for example, PTPRC, DEXI and ZFP36L1). PMID:26616563

  4. Meiosis gene inventory of four ciliates reveals the prevalence of a synaptonemal complex-independent crossover pathway.

    Science.gov (United States)

    Chi, Jingyun; Mahé, Frédéric; Loidl, Josef; Logsdon, John; Dunthorn, Micah

    2014-03-01

    To establish which meiosis genes are present in ciliates, and to look for clues as to which recombination pathways may be treaded by them, four genomes were inventoried for 11 meiosis-specific and 40 meiosis-related genes. We found that the set of meiosis genes shared by Tetrahymena thermophila, Paramecium tetraurelia, Ichthyophthirius multifiliis, and Oxytricha trifallax is consistent with the prevalence of a Mus81-dependent class II crossover pathway that is considered secondary in most model eukaryotes. There is little evidence for a canonical class I crossover pathway that requires the formation of a synaptonemal complex (SC). This gene inventory suggests that meiotic processes in ciliates largely depend on mitotic repair proteins for executing meiotic recombination. We propose that class I crossovers and SCs were reduced sometime during the evolution of ciliates. Consistent with this reduction, we provide microscopic evidence for the presence only of degenerate SCs in Stylonychia mytilus. In addition, lower nonsynonymous to synonymous mutation rates of some of the meiosis genes suggest that, in contrast to most other nuclear genes analyzed so far, meiosis genes in ciliates are largely evolving at a slower rate than those genes in fungi and animals.

  5. Revealing a Ring-like Cluster Complex in a Tidal Tail of the Starburst Galaxy NGC 2146

    CERN Document Server

    Adamo, A; Gallagher, J S; Bastian, N; Ryon, J; Westmoquette, M S; Konstantopoulos, I S; Zackrisson, E; Larsen, S S; Silva-Villa, E; Charlton, J C; Weisz, D R

    2012-01-01

    We report the discovery of a ring-like cluster complex in the starburst galaxy NGC 2146. The Ruby Ring, so named due to its appearance, shows a clear ring-like distribution of star clusters around a central object. It is located in one of the tidal streams which surround the galaxy. NGC 2146 is part of the Snapshot Hubble U-band Cluster Survey (SHUCS). The WFC3/F336W data has added critical information to the available archival Hubble Space Telescope imaging set of NGC 2146, allowing us to determine ages, masses, and extinctions of the clusters in the Ruby Ring. These properties have then been used to investigate the formation of this extraordinary system. We find evidence of a spatial and temporal correlation between the central cluster and the clusters in the ring. The latter are about 4 Myr younger than the central cluster, which has an age of 7 Myr. This result is supported by the H alpha emission which is strongly coincident with the ring, and weaker at the position of the central cluster. From the deriv...

  6. Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease.

    Directory of Open Access Journals (Sweden)

    Maria Wilbe

    2015-06-01

    Full Text Available The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA in patients' sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA(H and speckled ANA (ANA(S staining pattern, and also steroid-responsive meningitis-arteritis (SRMA are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAMTOR3, DDIT4L and PPP3CA located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.

  7. Crystal structures of PRK1 in complex with the clinical compounds lestaurtinib and tofacitinib reveal ligand induced conformational changes.

    Directory of Open Access Journals (Sweden)

    Philip Chamberlain

    Full Text Available Protein kinase C related kinase 1 (PRK1 is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

  8. The probabilistic niche model reveals the niche structure and role of body size in a complex food web.

    Science.gov (United States)

    Williams, Richard J; Anandanadesan, Ananthi; Purves, Drew

    2010-08-09

    The niche model has been widely used to model the structure of complex food webs, and yet the ecological meaning of the single niche dimension has not been explored. In the niche model, each species has three traits, niche position, diet position and feeding range. Here, a new probabilistic niche model, which allows the maximum likelihood set of trait values to be estimated for each species, is applied to the food web of the Benguela fishery. We also developed the allometric niche model, in which body size is used as the niche dimension. About 80% of the links in the empirical data are predicted by the probabilistic niche model, a significant improvement over recent models. As in the niche model, species are uniformly distributed on the niche axis. Feeding ranges are exponentially distributed, but diet positions are not uniformly distributed below the predator. Species traits are strongly correlated with body size, but the allometric niche model performs significantly worse than the probabilistic niche model. The best-fit parameter set provides a significantly better model of the structure of the Benguela food web than was previously available. The methodology allows the identification of a number of taxa that stand out as outliers either in the model's poor performance at predicting their predators or prey or in their parameter values. While important, body size alone does not explain the structure of the one-dimensional niche.

  9. De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.).

    Science.gov (United States)

    Xu, Liang; Wang, Yan; Liu, Wei; Wang, Jin; Zhu, Xianwen; Zhang, Keyun; Yu, Rugang; Wang, Ronghua; Xie, Yang; Zhang, Wei; Gong, Yiqin; Liu, Liwang

    2015-07-01

    Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops.

  10. The complex circumnuclear environment of the broad-line radio galaxy 3C 390.3 revealed by Chandra HETG

    CERN Document Server

    Tombesi, F; Kallman, T; Reynolds, C S; Mushotzky, R F; Braito, V; Behar, E; Leutenegger, M A; Cappi, M

    2016-01-01

    We present the first high spectral resolution X-ray observation of the broad-line radio galaxy 3C 390.3 obtained with the high energy transmission grating (HETG) spectrometer on board the Chandra X-ray Observatory. The spectrum shows complex emission and absorption features in both the soft X-rays and Fe K band. We detect emission and absorption lines in the energy range between E = 700-1000 eV associated with ionized Fe L transitions (Fe XVII-XX). An emission line at the energy of E=6.4 keV consistent with the Fe K\\alpha is also observed. Our best-fit model requires at least three different components: (i) a hot emission component likely associated with the hot interstellar medium in this elliptical galaxy with temperature kT=0.5+/-0.1 keV; (ii) a warm absorber with ionization parameter log\\xi=2.3+/-0.5 erg s^{-1} cm, column density logN_H=20.7+/-0.1 cm^{-2}, and outflow velocity of v_{out}<150 km s^{-1}; (iii) a lowly ionized reflection component in the Fe K band likely associated with the optical broad ...

  11. MAGIC reveals a complex morphology within the unidentified gamma-ray source HESS J1857+026

    CERN Document Server

    Aleksić, J; Antonelli, L A; Antoranz, P; Babic, A; Bangale, P; de Almeida, U Barres; Barrio, J A; González, J Becerra; Bednarek, W; Bernardini, E; Biland, A; Blanch, O; Bonnefoy, S; Bonnoli, G; Borracci, F; Bretz, T; Carmona, E; Carosi, A; Colin, P; Colombo, E; Contreras, J L; Cortina, J; Covino, S; Da Vela, P; Dazzi, F; De Angelis, A; De Caneva, G; De Lotto, B; Mendez, C Delgado; Doert, M; Domínguez, A; Prester, D Dominis; Dorner, D; Doro, M; Einecke, S; Eisenacher, D; Elsaesser, D; Farina, E; Ferenc, D; Carreto, D Fidalgo; Fonseca, M V; Font, L; Frantzen, K; Fruck, C; López, R J García; Garczarczyk, M; Terrats, D Garrido; Gaug, M; Godinović, N; Muñoz, A González; Gozzini, S R; Hadasch, D; Hayashida, M; Herrera, J; Herrero, A; Hildebrand, D; Hose, J; Hrupec, D; Idec, W; Kadenius, V; Kellermann, H; Kodani, K; Konno, Y; Krause, J; Kubo, H; Kushida, J; La Barbera, A; Lelas, D; Lewandowska, N; Lindfors, E; Lombardi, S; López, M; López-Coto, R; López-Oramas, A; Lorenz, E; Lozano, I; Makariev, M; Mallot, K; Maneva, G; Mankuzhiyil, N; Mannheim, K; Maraschi, L; Marcote, B; Mariotti, M; Martínez, M; Mazin, D; Menzel, U; Meucci, M; Miranda, J M; Mirzoyan, R; Moralejo, A; Munar-Adrover, P; Nakajima, D; Niedzwiecki, A; Nilsson, K; Nishijima, K; Noda, K; Nowak, N; Wilhelmi, E de Oña; Orito, R; Overkemping, A; Klepser, S; Paiano, S; Palatiello, M; Paneque, D; Paoletti, R; Paredes, J M; Paredes-Fortuny, X; Partini, S; Persic, M; Prada, F; Moroni, P G Prada; Prandini, E; Preziuso, S; Puljak, I; Reinthal, R; Rhode, W; Ribó, M; Rico, J; Garcia, J Rodriguez; Rügamer, S; Saggion, A; Saito, T; Saito, K; Satalecka, K; Scalzotto, V; Scapin, V; Schultz, C; Schweizer, T; Shore, S N; Sillanpää, A; Sitarek, J; Snidaric, I; Sobczynska, D; Spanier, F; Stamatescu, V; Stamerra, A; Steinbring, T; Storz, J; Strzys, M; Sun, S; Surić, T; Takalo, L; Takami, H; Tavecchio, F; Temnikov, P; Terzić, T; Tescaro, D; Teshima, M; Thaele, J; Tibolla, O; Torres, D F; Toyama, T; Treves, A; Uellenbeck, M; Vogler, P; Wagner, R M; Zandanel, F; Zanin, R

    2014-01-01

    HESS J1857+026 is an extended TeV gamma-ray source that was discovered by H.E.S.S. as part of its Galactic plane survey. Given its broadband spectral energy distribution and its spatial coincidence with the young energetic pulsar PSR J1856+024, the source has been put forward as a pulsar wind nebula (PWN) candidate. MAGIC has performed follow-up observations aimed at mapping the source down to energies approaching 100 GeV in order to better understand its complex morphology. HESS J1857+026 was observed by MAGIC in 2010, yielding 29 hours of good quality stereoscopic data that allowed us to map the source region in two separate ranges of energy. We present an energy spectrum of the region, which bridges the gap between the GeV emission measured by Fermi-LAT and the multi-TeV emission measured by H.E.S.S., together with a detailed analysis of its energy-dependent morphology. We couple these results with archival multi-wavelength data and outline evidence in favor of a two-source scenario, whereby one source is ...

  12. Taxonomic analysis of cryptococcus species complex strain S8012 revealed Cryptococcus gattii with high heterogeneity on the genetics

    Institute of Scientific and Technical Information of China (English)

    CHEN Min; LIAO Wan-qing; WU Shao-xi; YAO Zhi-rong; PAN Wei-hua; LIAO Yong

    2011-01-01

    Background Initially, Cryptococcus (C.) neoformans was previously divided into two varieties comprising C. neoformans var. neoformans and C. neoformans var. gattii. Currently, taxonomic studies defined C. neoformans as C. species complex, which contains C. neoformans var. neoformans (serotype D), the hybrid isolates (serotype AD), C. neoformans var. grubii (serotype A) and C. gattii (serotypes B and C). However, Liao and his team once isolated a unique C. gattii isolate, namely strain S8012 with unique phenotype from cerebrospinal fluid (CSF) of a 43-year-old male patient in the Shanghai Changzheng Hospital and described as C. neoformans var. shanghaiensis in 1980s. The aim of this study was to explore the genetic background and polymorphism of Chinese clinical C. gattii isolates.Methods S8012 was analyzed as representative strain using the M13-polymerase chain reaction (PCR) fingerprinting pattern and multilocus sequence analysis including internal transcribed spacers of rDNA (ITS region), the intergenic spacer 1 regions (IGS1), RPB1, RPB2, CNLAC1, and TEF1 genes.Results The PCR fingerprinting pattern results showed strain S8012 belonged to molecular types VGI, and phylogenetic analysis suggested strain S8012 was grouped into the cluster of C. gattii environmental isolates originated from Eucalyptus camaldulensis trees in Australia.Conclusion C. gattii isolates from Chinese patients expresses high polymorphism on the phenotype, and molecular type VQI isolates from China have a close genetic relationship with the C. gattii isolates from Australia.

  13. Phylogeography of North African Atlas cedar (Cedrus atlantica, Pinaceae): Combined molecular and fossil data reveal a complex Quaternary history.

    Science.gov (United States)

    Terrab, Anass; Hampe, Arndt; Lepais, Olivier; Talavera, Salvador; Vela, Errol; Stuessy, Tod F

    2008-10-01

    Northwest Africa is a major hotspot of plant biodiversity, but very little is known about the Quaternary range dynamics of plant species in this region. Here we investigate the range-wide population structure and phylogeography of Atlas cedar (Cedrus atlantica), an emblematic forest tree endemic to Morocco and Algeria. We genotyped 261 individuals from 11 populations using AFLP markers. Data were analyzed using both conventional F(ST)-based techniques and Bayesian clustering. Overall population differentiation was high (F(ST) = 0.25). Two major groups of populations were identified, one distributed through the Rif and Middle Atlas mountains in Morocco and the other through the Algerian Tell Atlas and Aurès mountains as well as the Middle Atlas. Combined molecular and fossil data indicate that C. atlantica survived the Last Glacial Maximum in at least three disjunct refugia along the coast of the Mediterranean Sea, whereas the Middle Atlas, today the core of the species range, has been colonized relatively recently (<10000 yr BP). The colonization history of individual populations has left clear imprints in their present-day diversity, which may vary greatly even between nearby stands. Our study illustrates how integrating different data sources and analytical approaches can help elucidate complex range dynamics that would otherwise remain undeciphered.

  14. The Human SepSecS-tRNA[superscript Sec] Complex Reveals the Mechanism of Selenocysteine Formation

    Energy Technology Data Exchange (ETDEWEB)

    Palioura, Sotiria; Sherrer, R. Lynn; Steitz, Thomas A.; Söll, Dieter; Simonovic, Miljan; (Yale); (UIC)

    2009-08-13

    Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNA{sup Sec} in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA{sup Sec} formation. Two tRNA{sup Sec} molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-T{Upsilon}C arm (where {Upsilon} indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA{sup Sec}, but not free phosphoserine, to be oriented properly for the reaction to occur.

  15. Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex

    Directory of Open Access Journals (Sweden)

    Walaa Oweis

    2016-09-01

    Full Text Available Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s. Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.

  16. Insights from genome of Clostridium butyricum INCQS635 reveal mechanisms to convert complex sugars for biofuel production.

    Science.gov (United States)

    Bruce, Thiago; Leite, Fernanda Gomes; Miranda, Milene; Thompson, Cristiane C; Pereira, Nei; Faber, Mariana; Thompson, Fabiano L

    2016-03-01

    Clostridium butyricum is widely used to produce organic solvents such as ethanol, butanol and acetone. We sequenced the entire genome of C. butyricum INCQS635 by using Ion Torrent technology. We found a high contribution of sequences assigned for carbohydrate subsystems (15-20 % of known sequences). Annotation based on protein-conserved domains revealed a higher diversity of glycoside hydrolases than previously found in C. acetobutylicum ATCC824 strain. More than 30 glycoside hydrolases (GH) families were found; families of GH involved in degradation of galactan, cellulose, starch and chitin were identified as most abundant (close to 50 % of all sequences assigned as GH) in C. butyricum INCQS635. KEGG metabolic pathways reconstruction allowed us to verify possible routes in the C. butyricum INCQS635 and C. acetobutylicum ATCC824 genomes. Metabolic pathways for ethanol synthesis are similar for both species, but alcohol dehydrogenase of C. butyricum INCQS635 and C. acetobutylicum ATCC824 was different. The genomic repertoire of C. butyricum is an important resource to underpin future studies towards improved solvents production.

  17. The Genome of the Obligately Intracellular Bacterium Ehrlichia canis Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies

    Energy Technology Data Exchange (ETDEWEB)

    Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Doyle, C Kuyler [Center for Biodenfense and Emerging Infectious Diseases; Lykidis, A [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Francino, M P [U.S. Department of Energy, Joint Genome Institute; Chain, Patrick S [ORNL; Shin, M [U.S. Department of Energy, Joint Genome Institute; Malfatti, Stephanie [Lawrence Livermore National Laboratory (LLNL); Larimer, Frank W [ORNL; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Detter, J C [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Richardson, P M [U.S. Department of Energy, Joint Genome Institute; Yu, X J [Center for Biodenfense and Emerging Infectious Diseases; Walker, D H [Center for Biodenfense and Emerging Infectious Diseases; McBride, J W [Center for Biodenfense and Emerging Infectious Diseases; Kyripides, N C [U.S. Department of Energy, Joint Genome Institute

    2006-01-01

    Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, {alpha}-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

  18. Reprogramming of Yersinia from virulent to persistent mode revealed by complex in vivo RNA-seq analysis.

    Directory of Open Access Journals (Sweden)

    Kemal Avican

    2015-01-01

    Full Text Available We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.

  19. Revealing the complex nature of the strong gravitationally lensed system H-ATLAS J090311.6+003906 using ALMA

    CERN Document Server

    Dye, S; Swinbank, A M; Vlahakis, C; Nightingale, J W; Dunne, L; Eales, S A; Smail, Ian; Oteo-Gomez, I; Hunter, T; Negrello, M; Dannerbauer, H; Ivison, R J; Gavazzi, R; Cooray, A; van der Werf, P

    2015-01-01

    We have modelled Atacama Large Millimeter/sub-millimeter Array (ALMA) long baseline imaging of the strong gravitational lens system H-ATLAS J090311.6+003906 (SDP.81). We have reconstructed the distribution of continuum emission in the z=3.042 source and we have determined its kinematic properties by reconstructing CO line emission. The continuum imaging reveals a highly non-uniform distribution of dust with clumps on scales of ~200pc. In contrast, the CO line emission shows a relatively smooth velocity field which resembles disk-like dynamics. Modelling the velocity field as a rotating disk indicates an inclination angle of (40 +/- 5) degrees, implying an intrinsic asymptotic rotation velocity of 320km/s and a dynamical mass of 3.5x10^{10} M_sol within 1.5kpc. We obtain similar estimates of the total molecular gas mass of 2.7x10^{10} M_sol and 1.4x10^{10} M_sol from the dust continuum emission and CO emission respectively. Our new reconstruction of the lensed HST near-infrared emission shows two objects that ...

  20. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  1. Digitonthophagus Balthasar, 1959: taxonomy, systematics, and morphological phylogeny of the genus revealing an African species complex (Coleoptera: Scarabaeidae: Scarabaeinae).

    Science.gov (United States)

    Génier, François; Moretto, Philippe

    2017-03-31

    The taxonomy and systematics of the genus Digitonthophagus Balthasar (Coleoptera: Scarabaeidae: Scarabaeinae: Onthophagini) is revised. A detailed study of the male genitalia combined with external morphology suggests that the variability, previously recognized, for D. gazella is hiding a species complex within the Afrotropical region and the Arabian Peninsula. The current study recognizes 16 species; 13 from the Afrotropical region and Arabian Peninsula and three from the eastern portion of the Saharo-Arabian region and the continental Indomalayan region. Species are organized into six species groups based on the results of the morphology-based phylogenetic analysis. The following 12 species are described as new: D. aksumensis Génier new species; D. biflagellatus Génier new species; D. dilatatus Génier new species; D. eucatta Génier new species; D. falciger Génier new species; D. fimator Génier new species; D. namaquensis Génier new species; D. petilus Génier new species; D. sahelicus Moretto new species; D. uks Génier new species; D. ulcerosus Génier new species; and D. viridicollis Génier new species. In order to stabilize nomenclature, lectotypes are designated for Scarabaeus bonasus Fabricius, 1775; Scarabaeus catta Fabricius, 1787, and Onthophagus gazella lusinganus d'Orbigny. A neotype is designated for Scarabaeus dorcas Olivier, 1789 whose status and synonymy need to be altered in order to clarify the status of Scarabaeus gazella auctorum, the widely introduced species with economic importance. A naming scheme is presented for the sclerites of the internal sac. External and male genitalia are illustrated and distribution maps are provided for each species.

  2. Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes

    Directory of Open Access Journals (Sweden)

    Takashi Hamaji

    2016-05-01

    Full Text Available Sex-determining regions (SDRs or mating-type (MT loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT–, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale–V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

  3. Transcriptional Profiling of Vibrio parahaemolyticus exsA reveals a complex activation network for type III secretion

    Directory of Open Access Journals (Sweden)

    Aaron C. Liu

    2015-10-01

    Full Text Available Vibrio parahaemolyticus (Vp is a marine halophilic bacterium that is commonly associated with oysters and shrimp. Human consumption of contaminated shellfish can result in Vp mediated gastroenteritis and severe diarrheal disease. Vp encodes two type 3 secretion systems (T3SS-I and T3SS-II that have been functionally implicated in cytotoxicity and enterotoxicity respectively. In this study, we profiled protein secretion and temporal promoter activities associated with exsA and exsB gene expression. exsA is an AraC-like transcriptional activator that is critical for activating multiple operons that encode T3SS-1 genes, whereas exsB is thought to encode an outer membrane pilotin component for T3SS-1. The exsBA genetic locus has two predicted promoter elements. The predicted exsB and exsA promoters were individually cloned upstream of luxCDABE genes in reporter plasmid constructs allowing for in situ, real-time quantitative light emission measurements under many growth conditions. Low calcium growth conditions supported maximal exsB and exsA promoter activation. exsB promoter activity exhibited high basal activity and resulted in an exsBA co-transcript. Furthermore, a separate proximal exsA promoter showed initial low basal activity yet eventually exceeded that of exsB and reached maximal levels after 2.5 hours corresponding to an entry into early log phase. exsA promoter activity was significantly higher at 30oC than 37oC, which also coincided with increased secretion levels of specific T3SS-1 effector proteins. Lastly, bioinformatic analyses identified a putative expanded ExsA binding motif for multiple transcriptional operons. These findings suggest a two wave model of Vp T3SS-I induction that integrates two distinct promoter elements and environmental signals into a complex ExsA activation framework.

  4. Phylogenetic analysis of glycerol 3-phosphate acyltransferases in opisthokonts reveals unexpected ancestral complexity and novel modern biosynthetic components.

    Directory of Open Access Journals (Sweden)

    Heather C Smart

    Full Text Available Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT, have been described, and characterized primarily in model organisms like Saccharomyces cerevisiae and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of 'fungal' orthologs in the basal taxa of the holozoa and 'animal' orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens.

  5. Bayesian inference of phylogeny, morphology and range evolution reveals a complex evolutionary history in St. John's wort (Hypericum).

    Science.gov (United States)

    Meseguer, Andrea Sánchez; Aldasoro, Juan Jose; Sanmartín, Isabel

    2013-05-01

    The genus Hypericum L. ("St. John's wort", Hypericaceae) comprises nearly 500 species of shrubs, trees and herbs distributed mainly in temperate regions of the Northern Hemisphere, but also in high-altitude tropical and subtropical areas. Until now, molecular phylogenetic hypotheses on infra-generic relationships have been based solely on the nuclear marker ITS. Here, we used a full Bayesian approach to simultaneously reconstruct phylogenetic relationships, divergence times, and patterns of morphological and range evolution in Hypericum, using nuclear (ITS) and plastid DNA sequences (psbA-trnH, trnS-trnG, trnL-trnF) of 186 species representing 33 of the 36 described morphological sections. Consistent with other studies, we found that corrections of the branch length prior helped recover more realistic branch lengths in by-gene partitioned Bayesian analyses, but the effect was also seen within single genes if the overall mutation rate differed considerably among sites or regions. Our study confirms that Hypericum is not monophyletic with the genus Triadenum embedded within, and rejects the traditional infrageneric classification, with many sections being para- or polyphyletic. The small Western Palearctic sections Elodes and Adenotrias are the sister-group of a geographic dichotomy between a mainly New World clade and a large Old World clade. Bayesian reconstruction of morphological character states and range evolution show a complex pattern of morphological plasticity and inter-continental movement within the genus. The ancestors of Hypericum were probably tropical shrubs that migrated from Africa to the Palearctic in the Early Tertiary, concurrent with the expansion of tropical climates in northern latitudes. Global climate cooling from the Mid Tertiary onwards might have promoted adaptation to temperate conditions in some lineages, such as the development of the herbaceous habit or unspecialized corollas.

  6. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  7. The complex arrangement of an "aorto-jejunal paraduodenal" fossa, as revealed by dissection of human posterior parietal peritoneum.

    Science.gov (United States)

    Barberini, Fabrizio; Zani, Augusto; Ripani, Maurizio; Di Nitto, Valentina; Brunone, Francesca

    2007-01-01

    Peritoneal fossae derive from normal or anomalous coalescence of the peritoneum during fetal development, or from the course of retroperitoneal vessels. Clinically, internal abdominal hernias may be housed inside these fossae. In this report from an autopsy, a singular peritoneal fossa was delimited superiorly by an arcuate serous fold, raised up by the inferior mesenteric vein, and infero-posteriorly by two (right and left) avascular folds, extending from the abdominal aorta to the jejunum. The right fold reached the duodeno-jejunal flexure, which was located on the right side of the aorta. The left fold subdivided into two, anterior and posterior, secondary folds. The anterior fold reached the superior edge of the first jejunal loop, and the posterior fold turned medially to connect with the inferior edge of the proximal limb of the same loop. This fossa consisted of three recesses: superior, Located behind the subserous vascular arch, antero-inferior and postero-inferior, separated by interposition of the left posterior secondary fold, between the jejunum and aorta. The complex arrangement of this fossa suggests that it might have originated from a coalescence arising beyond the duodeno-jejunal flexure and including the first jejunal loop, and from the subserous course of the inferior mesenteric vein. Because of displacement to the right of the flexure, processes of coalescence in a location normally occupied by the ascending duodenum might have occurred in a similar pattern for the jejunum, involving the mesoduodenum and the proximal part of the mesentery. Labyrinthine fossae like this might cause strangulation of internal abdominal hernias and hinder intraoperative maneuvers.

  8. High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment

    Energy Technology Data Exchange (ETDEWEB)

    Handley, Kim M.; Wrighton, Kelly E.; Piceno, Y. M.; Anderson, Gary L.; DeSantis, Todd; Williams, Kenneth H.; Wilkins, Michael J.; N' Guessan, A. L.; Peacock, Aaron; Bargar, John R.; Long, Philip E.; Banfield, Jillian F.

    2012-06-13

    There is increasing interest in harnessing the functional diversity of indigenous microbial communities to transform and remediate a wide range of environmental contaminants. Understanding the response of communities to stimulation, including flanking taxa, presents important opportunities for optimizing remediation approaches. We used high-density PhyloChip microarray analysis to comprehensively determine community membership and abundance patterns amongst a suite of samples from U(VI) bioremediation experiments. Samples were unstimulated or collected during Fe(III) and sulfate reduction from an acetate-augmented aquifer in Rifle, Colorado, and from laboratory experiments using field-collected materials. Results showed the greatest diversity in abundant SRB lineages was present in naturally-reduced sediment. Desulfuromonadales and Desulfobacterales were consistently identified as the dominant Fe(III)- and sulfate-reducing bacteria (IRB and SRB) throughout acetate amendment experiments. Stimulated communities also exhibited a high degree of functional redundancy amongst enriched flanking members. Not surprisingly, competition for both sulfate and iron was evident amongst abundant taxa, but the distribution and abundance of these ancillary SRB (Peptococcaceae, Desulfovibrionales and Syntrophobacterales), and lineages containing IRB (excluding Desulfobacteraceae) was heterogeneous amongst sample types. Interesting, amongst the most abundant taxa, particularly during sulfate reduction, were Epsilonproteobacteria that perform microaerobic or nitrate-dependant sulfur oxidation, and a number of bacteria other than Geobacteraceae that may enzymatically reduce U(VI). Finally, in depth community probing with PhyloChip determined the efficacy of experimental approaches, notably revealing striking similarity amongst stimulated sediment (from drill cores and in-situ columns) and groundwater communities, and demonstrating that sediment-packed in-situ (down-well) columns served

  9. K2P2.1 (TREK-1)–activator complexes reveal a cryptic selectivity filter binding site

    Energy Technology Data Exchange (ETDEWEB)

    Lolicato, Marco; Arrigoni, Cristina; Mori, Takahiro; Sekioka, Yoko; Bryant, Clifford; Clark, Kimberly A.; Minor, Jr , Daniel L. (Ono); (UCSF)

    2017-07-10

    Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses1, 2, 3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit4, 5, 6. In contrast to other potassium channels, K2P channels use a selectivity filter ‘C-type’ gate7, 8, 9, 10 as the principal gating site. Despite recent advances3, 11, 12, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators—an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation–π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

  10. Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

    Science.gov (United States)

    Gioannini, Theresa L; Teghanemt, Athmane; Zhang, DeSheng; Esparza, Gregory; Yu, Liping; Weiss, Jerrold

    2014-08-01

    A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

  11. Pentacoordinate and Hexacoordinate Mn(III) Complexes of Tetradentate Schiff-Base Ligands Containing Tetracyanidoplatinate(II) Bridges and Revealing Uniaxial Magnetic Anisotropy.

    Science.gov (United States)

    Nemec, Ivan; Herchel, Radovan; Trávníček, Zdeněk

    2016-12-08

    Crystal structures and magnetic properties of polymeric and trinuclear heterobimetallic Mn(III)···Pt(II)···Mn(III) coordination compounds, prepared from the Ba[Pt(CN)₄] and [Mn(L4A/B)(Cl)] (1a/b) precursor complexes, are reported. The polymeric complex [{Mn(L4A)}₂{μ⁴-Pt(CN)₄}]n (2a), where H₂L4A = N,N'-ethylene-bis(salicylideneiminate), comprises the {Mn(L4A)} moieties covalently connected through the [Pt(CN)₄](2-) bridges, thus forming a square-grid polymeric structure with the hexacoordinate Mn(III) atoms. The trinuclear complex [{Mn(L4B)}₂{μ-Pt(CN)₄}] (2b), where H₂L4B = N,N'-benzene-bis(4-aminodiethylene-salicylideneiminate), consists of two [{Mn(L4B)} moieties, involving pentacoordinate Mn(III) atoms, bridged through the tetracyanidoplatinate (II) bridges to which they are coordinated in a trans fashion. Both complexes possess uniaxial type of magnetic anisotropy, with D (the axial parameter of zero-field splitting) = -3.7(1) in 2a and -2.2(1) cm(-1) in 2b. Furthermore, the parameters of magnetic anisotropy 2a and 2b were also thoroughly studied by theoretical complete active space self-consistent field (CASSCF) methods, which revealed that the former is much more sensitive to the ligand field strength of the axial ligands.

  12. Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America.

    Science.gov (United States)

    Perez, Manolo F; Carstens, Bryan C; Rodrigues, Gustavo L; Moraes, Evandro M

    2016-02-01

    The Pilosocereus aurisetus complex consists of eight cactus species with a fragmented distribution associated to xeric enclaves within the Cerrado biome in eastern South America. The phylogeny of these species is incompletely resolved, and this instability complicates evolutionary analyses. Previous analyses based on both plastid and microsatellite markers suggested that this complex contained species with inherent phylogeographic structure, which was attributed to recent diversification and recurring range shifts. However, limitations of the molecular markers used in these analyses prevented some questions from being properly addressed. In order to better understand the relationship among these species and make a preliminary assessment of the genetic structure within them, we developed anonymous nuclear loci from pyrosequencing data of 40 individuals from four species in the P. aurisetus complex. The data obtained from these loci were used to identify genetic clusters within species, and to investigate the phylogenetic relationship among these inferred clusters using a species tree methodology. Coupled with a palaeodistributional modelling, our results reveal a deep phylogenetic and climatic disjunction between two geographic lineages. Our results highlight the importance of sampling more regions from the genome to gain better insights on the evolution of species with an intricate evolutionary history. The methodology used here provides a feasible approach to develop numerous genealogical molecular markers throughout the genome for non-model species. These data provide a more robust hypothesis for the relationship among the lineages of the P. aurisetus complex.

  13. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  14. Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

    Directory of Open Access Journals (Sweden)

    An Gynheung

    2011-04-01

    Full Text Available Abstract Background Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS and sequencing-by-synthesis (SBS. Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield, LaGrue (low milling yield, Ilpumbyeo (high eating quality, YR15965 (low eating quality, and Nipponbare (control. Results The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90, and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved cis regulatory elements were identified. Numerous specifically expressed transcription factor (TF genes were identified in Cypress (282, LaGrue (312, Ilpumbyeo (363, YR15965 (260, and Nipponbare (357. Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase and granule bound starch synthase I (GBSS I in Cypress than that in LaGrue during early seed development. Conclusion This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved

  15. Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics

    Directory of Open Access Journals (Sweden)

    Symula Rebecca E

    2013-02-01

    Full Text Available Abstract Background Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI. This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep. Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60. Results MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945. We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16. Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non

  16. Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

    NARCIS (Netherlands)

    Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

    1996-01-01

    We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstr

  17. Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

    Directory of Open Access Journals (Sweden)

    A.S.G. Lima

    2002-01-01

    Full Text Available PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic and ERIC (Enterobacterial Repetitive Intergenic Consensus sequences (rep-PCR found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

  18. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    Energy Technology Data Exchange (ETDEWEB)

    Rockel, Beate [Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); Schmaler, Tilo; Huang, Xiaohua [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany); Dubiel, Wolfgang, E-mail: Wolfgang.dubiel@charite.de [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany)

    2014-07-25

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  19. Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

    Energy Technology Data Exchange (ETDEWEB)

    Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P [TAM

    2012-08-01

    Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a kcat/KM higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest kcat/KM of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.

  20. Transcriptome analysis of a rotenone model of parkinsonism reveals complex I-tied and -untied toxicity mechanisms common to neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Yofre Cabeza-Arvelaiz

    Full Text Available The pesticide rotenone, a neurotoxin that inhibits the mitochondrial complex I, and destabilizes microtubules (MT has been linked to Parkinson disease (PD etiology and is often used to model this neurodegenerative disease (ND. Many of the mechanisms of action of rotenone are posited mechanisms of neurodegeneration; however, they are not fully understood. Therefore, the study of rotenone-affected functional pathways is pertinent to the understanding of NDs pathogenesis. This report describes the transcriptome analysis of a neuroblastoma (NB cell line chronically exposed to marginally toxic and moderately toxic doses of rotenone. The results revealed a complex pleiotropic response to rotenone that impacts a variety of cellular events, including cell cycle, DNA damage response, proliferation, differentiation, senescence and cell death, which could lead to survival or neurodegeneration depending on the dose and time of exposure and cell phenotype. The response encompasses an array of physiological pathways, modulated by transcriptional and epigenetic regulatory networks, likely activated by homeostatic alterations. Pathways that incorporate the contribution of MT destabilization to rotenone toxicity are suggested to explain complex I-independent rotenone-induced alterations of metabolism and redox homeostasis. The postulated mechanisms involve the blockage of mitochondrial voltage-dependent anions channels (VDACs by tubulin, which coupled with other rotenone-induced organelle dysfunctions may underlie many presumed neurodegeneration mechanisms associated with pathophysiological aspects of various NDs including PD, AD and their variant forms. Thus, further investigation of such pathways may help identify novel therapeutic paths for these NDs.

  1. Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β-amyloid peptide revealed by affinity mass spectrometry and molecular modeling.

    Science.gov (United States)

    Maftei, Madalina; Tian, Xiaodan; Manea, Marilena; Exner, Thomas E; Schwanzar, Daniel; von Arnim, Christine A F; Przybylski, Michael

    2012-06-01

    Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.

  2. Pentacoordinate and Hexacoordinate Mn(III Complexes of Tetradentate Schiff-Base Ligands Containing Tetracyanidoplatinate(II Bridges and Revealing Uniaxial Magnetic Anisotropy

    Directory of Open Access Journals (Sweden)

    Ivan Nemec

    2016-12-01

    Full Text Available Crystal structures and magnetic properties of polymeric and trinuclear heterobimetallic MnIII···PtII···MnIII coordination compounds, prepared from the Ba[Pt(CN4] and [Mn(L4A/B(Cl] (1a/b precursor complexes, are reported. The polymeric complex [{Mn(L4A}2{μ4-Pt(CN4}]n (2a, where H2L4A = N,N’-ethylene-bis(salicylideneiminate, comprises the {Mn(L4A} moieties covalently connected through the [Pt(CN4]2− bridges, thus forming a square-grid polymeric structure with the hexacoordinate MnIII atoms. The trinuclear complex [{Mn(L4B}2{μ-Pt(CN4}] (2b, where H2L4B = N,N’-benzene-bis(4-aminodiethylene-salicylideneiminate, consists of two [{Mn(L4B} moieties, involving pentacoordinate MnIII atoms, bridged through the tetracyanidoplatinate (II bridges to which they are coordinated in a trans fashion. Both complexes possess uniaxial type of magnetic anisotropy, with D (the axial parameter of zero-field splitting = −3.7(1 in 2a and −2.2(1 cm−1 in 2b. Furthermore, the parameters of magnetic anisotropy 2a and 2b were also thoroughly studied by theoretical complete active space self-consistent field (CASSCF methods, which revealed that the former is much more sensitive to the ligand field strength of the axial ligands.

  3. Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

    Directory of Open Access Journals (Sweden)

    Zadeh Soheila

    2010-07-01

    Full Text Available Abstract Background HER2 gene copy status, and concomitant administration of trastuzumab (Herceptin, remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC and fluorescence in situ hybridization (FISH are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods. Methods In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms. Results Array-based comparative genomic hybridization (array CGH analysis of chromosome 17 resolved HER2 gene status in [20/20] (100% of cases and revealed additional chromosome 17 copy number changes in [18/20] (90% of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability. Conclusions These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17

  4. Energy transfer pathways in light-harvesting complexes of purple bacteria as revealed by global kinetic analysis of two-dimensional transient spectra.

    Science.gov (United States)

    Ostroumov, Evgeny E; Mulvaney, Rachel M; Anna, Jessica M; Cogdell, Richard J; Scholes, Gregory D

    2013-09-26

    Excited state dynamics in LH2 complexes of two purple bacterial species were studied by broad-band two-dimensional electronic spectroscopy. The optical response was measured in the 500-600 nm spectral region on the 0-400 fs time scale. Global target analysis of two-dimensional (2D) transient spectra revealed the main energy transfer pathways between carotenoid S2, 1Bu(-) and S1 states and bacteriochlorophyll Qx state. Global analysis ascertained the evolutionary and vibration-associated spectra, which also indicated the presence of a higher-lying vibrational level in the carotenoid S1 state. The estimation of the spectral overlap between the 1Bu(-) state and the Qx state indicated a significant contribution of the 1Bu(-) state to the overall S2-to-Qx excitation energy transfer.

  5. Mass spectrometric proteomics reveals that nuclear protein positive cofactor PC4 selectively binds to cross-linked DNA by a trans-platinum anticancer complex.

    Science.gov (United States)

    Du, Zhifeng; Luo, Qun; Yang, Liping; Bing, Tao; Li, Xianchan; Guo, Wei; Wu, Kui; Zhao, Yao; Xiong, Shaoxiang; Shangguan, Dihua; Wang, Fuyi

    2014-02-26

    An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a trans-platinum anticancer complex, trans-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by trans-PtTz.

  6. Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes.

    Science.gov (United States)

    Ciferri, Claudio; Chandramouli, Sumana; Donnarumma, Danilo; Nikitin, Pavel A; Cianfrocco, Michael A; Gerrein, Rachel; Feire, Adam L; Barnett, Susan W; Lilja, Anders E; Rappuoli, Rino; Norais, Nathalie; Settembre, Ethan C; Carfi, Andrea

    2015-02-10

    Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.

  7. Transcriptomic responses of a simplified soil microcosm to a plant pathogen and its biocontrol agent reveal a complex reaction to harsh habitat.

    Science.gov (United States)

    Perazzolli, Michele; Herrero, Noemí; Sterck, Lieven; Lenzi, Luisa; Pellegrini, Alberto; Puopolo, Gerardo; Van de Peer, Yves; Pertot, Ilaria

    2016-10-27

    Soil microorganisms are key determinants of soil fertility and plant health. Soil phytopathogenic fungi are one of the most important causes of crop losses worldwide. Microbial biocontrol agents have been extensively studied as alternatives for controlling phytopathogenic soil microorganisms, but molecular interactions between them have mainly been characterised in dual cultures, without taking into account the soil microbial community. We used an RNA sequencing approach to elucidate the molecular interplay of a soil microbial community in response to a plant pathogen and its biocontrol agent, in order to examine the molecular patterns activated by the microorganisms. A simplified soil microcosm containing 11 soil microorganisms was incubated with a plant root pathogen (Armillaria mellea) and its biocontrol agent (Trichoderma atroviride) for 24 h under controlled conditions. More than 46 million paired-end reads were obtained for each replicate and 28,309 differentially expressed genes were identified in total. Pathway analysis revealed complex adaptations of soil microorganisms to the harsh conditions of the soil matrix and to reciprocal microbial competition/cooperation relationships. Both the phytopathogen and its biocontrol agent were specifically recognised by the simplified soil microcosm: defence reaction mechanisms and neutral adaptation processes were activated in response to competitive (T. atroviride) or non-competitive (A. mellea) microorganisms, respectively. Moreover, activation of resistance mechanisms dominated in the simplified soil microcosm in the presence of both A. mellea and T. atroviride. Biocontrol processes of T. atroviride were already activated during incubation in the simplified soil microcosm, possibly to occupy niches in a competitive ecosystem, and they were not further enhanced by the introduction of A. mellea. This work represents an additional step towards understanding molecular interactions between plant pathogens and biocontrol

  8. Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability

    Energy Technology Data Exchange (ETDEWEB)

    Hansman, Grant S.; Biertümpfel, Christian; Georgiev, Ivelin; McLellan, Jason S.; Chen, Lei; Zhou, Tongqing; Katayama, Kazuhiko; Kwong, Peter D. (NIH); (NIID-Japan)

    2011-10-10

    Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal {alpha}fucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical {alpha}fucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.

  9. Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2 emitting source under conditions of nutrient overload

    Science.gov (United States)

    Fisher-Wellman, Kelsey H.; Gilliam, Laura A. A.; Lin, Chien-Te; Cathey, Brook L.; Lark, Daniel S.; Neufer, P. Darrell

    2014-01-01

    Once regarded as “byproducts” of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, delineation of the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione depleting agent 1-chloro-2,4-dinitrobenzene (CDNB) led to a dose-dependent increase in pyruvate-supported JH2O2 emission, with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool following high fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increase the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status. PMID:24056031

  10. Volcanic deformation of Atosanupuri volcanic complex in the Kussharo caldera, Japan, from 1993 to 2016 revealed by JERS-1, ALOS, and ALOS-2 radar interferometry

    Science.gov (United States)

    Fujiwara, Satoshi; Murakami, Makoto; Nishimura, Takuya; Tobita, Mikio; Yarai, Hiroshi; Kobayashi, Tomokazu

    2017-06-01

    A series of uplifts and subsidences of a volcanic complex in the Kussharo caldera in eastern Hokkaido (Japan) has been revealed by interferometric analysis using archived satellite synthetic aperture radar data. A time series of interferograms from 1993 to 1998 showed the temporal evolution of a ground deformation process. The horizontal dimension of the deformation field was about 10 km in diameter, and the maximum amplitude of the deformation was >20 cm. Uplift started in 1994, and concurrent earthquake swarm activity was observed around the uplift area; however, no other phenomena were observed during this period. A subsidence process then followed, with the shape of the deformation forming a mirror image of the uplift. Model simulations suggest deformation was caused by a source at the depth of about 6 km and that the position of the source remained static throughout the episode. Subsidence of the volcanic complex was also observed by another satellite from 2007 to 2010, and likely continued for more than 10 years. In addition to the main uplift-subsidence sequence, small deformation patterns with short spatial wavelengths were observed at the center of the deforming area. Data from three satellites recorded small-scale subsidence of the Atosanupuri and Rishiri lava domes at a constant rate of approx. 1 cm/year from 1993 to 2016.[Figure not available: see fulltext.

  11. The Crystal Structure of BRAF in Complex with an Organoruthenium Inhibitor Reveals a Mechanism for Inhibition of an Active Form of BRAF Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Peng; Streu, Craig; Qin, Jie; Bregman, Howard; Pagano, Nicholas; Meggers, Eric; Marmorstein, Ronen (Wistar); (UPENN)

    2012-06-19

    Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF{sup V600E}, have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF{sup V600E} oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF{sup V600E} oncogene selective BRAF inhibitors for therapeutic application.

  12. Early evolution of large micro-organisms with cytological complexity revealed by microanalyses of 3.4 Ga organic-walled microfossils.

    Science.gov (United States)

    Sugitani, K; Mimura, K; Takeuchi, M; Lepot, K; Ito, S; Javaux, E J

    2015-11-01

    The Strelley Pool Formation (SPF) is widely distributed in the East Pilbara Terrane (EPT) of the Pilbara Craton, Western Australia, and represents a Paleoarchean shallow-water to subaerial environment. It was deposited ~3.4 billion years ago and displays well-documented carbonate stromatolites. Diverse putative microfossils (SPF microfossils) were recently reported from several localities in the East Strelley, Panorama, Warralong, and Goldsworthy greenstone belts. Thus, the SPF provides unparalleled opportunities to gain insights into a shallow-water to subaerial ecosystem on the early Earth. Our new micro- to nanoscale ultrastructural and microchemical studies of the SPF microfossils show that large (20-70 μm) lenticular organic-walled flanged microfossils retain their structural integrity, morphology, and chain-like arrangements after acid (HF-HCl) extraction (palynology). Scanning and transmitted electron microscopy of extracted microfossils revealed that the central lenticular body is either alveolar or hollow, and the wall is continuous with the surrounding smooth to reticulated discoidal flange. These features demonstrate the evolution of large micro-organisms able to form an acid-resistant recalcitrant envelope or cell wall with complex morphology and to form colonial chains in the Paleoarchean era. This study provides evidence of the evolution of very early and remarkable biological innovations, well before the presumed late emergence of complex cells.

  13. Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution.

    Science.gov (United States)

    Vo, Uybach; Vajpai, Navratna; Embrey, Kevin J; Golovanov, Alexander P

    2016-07-14

    The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos.

  14. Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5.

    Directory of Open Access Journals (Sweden)

    Garry Jason Forse

    Full Text Available The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.

  15. Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

    Science.gov (United States)

    Mühlbacher, Wolfgang; Mayer, Andreas; Sun, Mai; Remmert, Michael; Cheung, Alan C M; Niesser, Jürgen; Soeding, Johannes; Cramer, Patrick

    2015-10-01

    CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

  16. Musical rhythm and pitch: A differential effect on auditory dynamics as revealed by the N1/MMN/P3a complex.

    Science.gov (United States)

    Lelo-de-Larrea-Mancera, E Sebastian; Rodríguez-Agudelo, Yaneth; Solís-Vivanco, Rodolfo

    2017-06-01

    Music represents a complex form of human cognition. To what extent our auditory system is attuned to music is yet to be clearly understood. Our principal aim was to determine whether the neurophysiological operations underlying pre-attentive auditory change detection (N1 enhancement (N1e)/Mismatch Negativity (MMN)) and the subsequent involuntary attentional reallocation (P3a) towards infrequent sound omissions, are influenced by differences in musical content. Specifically, we intended to explore any interaction effects that rhythmic and pitch dimensions of musical organization may have over these processes. Results showed that both the N1e and MMN amplitudes were differentially influenced by rhythm and pitch dimensions. MMN latencies were shorter for musical structures containing both features. This suggests some neurocognitive independence between pitch and rhythm domains, but also calls for further address on possible interactions between both of them at the level of early, automatic auditory detection. Furthermore, results demonstrate that the N1e reflects basic sensory memory processes. Lastly, we show that the involuntary switch of attention associated with the P3a reflects a general-purpose mechanism not modulated by musical features. Altogether, the N1e/MMN/P3a complex elicited by infrequent sound omissions revealed evidence of musical influence over early stages of auditory perception. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

    Science.gov (United States)

    Elsworth, Brendan; Sanders, Paul R; Nebl, Thomas; Batinovic, Steven; Kalanon, Ming; Nie, Catherine Q; Charnaud, Sarah C; Bullen, Hayley E; de Koning Ward, Tania F; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2016-11-01

    The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

  18. Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium.

    Science.gov (United States)

    Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil

    2013-01-01

    In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

  19. Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

    Directory of Open Access Journals (Sweden)

    Saoussen Ben Khedher

    2013-09-01

    Full Text Available In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L-1 starch, 30 g L-1 soya bean and 9g L-1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch. Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

  20. Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.

    Science.gov (United States)

    Fisher-Wellman, Kelsey H; Gilliam, Laura A A; Lin, Chien-Te; Cathey, Brook L; Lark, Daniel S; Neufer, P Darrell

    2013-12-01

    Once regarded as a "by-product" of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, the sources and mechanisms regulating increased peroxide burden remain poorly defined and as such are difficult to target using pharmacotherapy. Here we identify the pyruvate dehydrogenase complex (PDC) as a key source of H2O2 within skeletal muscle mitochondria under conditions of depressed glutathione redox buffering integrity. Treatment of permeabilized myofibers with varying concentrations of the glutathione-depleting agent 1-chloro-2,4-dinitrobenzene led to a dose-dependent increase in pyruvate-supported JH2O2 emission (the flux of H2O2 diffusing out of the mitochondrial matrix into the surrounding assay medium), with emission rates eventually rising to exceed those of all substrate combinations tested. This striking sensitivity to glutathione depletion was observed in permeabilized fibers prepared from multiple species and was specific to PDC. Physiological oxidation of the cellular glutathione pool after high-fat feeding in rodents was found to elevate PDC JH2O2 emission, as well as increasing the sensitivity of the complex to GSH depletion. These findings reveal PDC as a potential major site of H2O2 production that is extremely sensitive to mitochondrial glutathione redox status. Published by Elsevier Inc.

  1. Complex patterns of population genetic structure of moose, Alces alces, after recent spatial expansion in Poland revealed by sex-linked markers.

    Science.gov (United States)

    Swisłocka, Magdalena; Czajkowska, Magdalena; Duda, Norbert; Danyłow, Jan; Owadowska-Cornil, Edyta; Ratkiewicz, Mirosław

    2013-01-01

    In recent years, human activity directly and indirectly influenced the demography of moose in Poland. The species was close to extinction, and only a few isolated populations survived after the Second World War; then, unprecedented demographic and spatial expansions had occurred, possibly generating a very complex pattern of population genetic structure at the present-day margins of the species range in Poland. Over 370 moose from seven populations were collected from Poland, and partial sequences of the mitochondrial control region (mtDNA-cr; 607 bp) were obtained. In addition, the entire mtDNA cytochrome b gene (1,140 bp) and Y-chromosome markers (1,982 bp in total) were studied in a chosen set of individuals. Twelve mtDNA haplotypes that all belonged to the European moose phylogroup were recorded. They could be divided into two distinct clades: Central Europe and the Ural Mountains. The first clade consists of three distinct groups/branches: Biebrza, Polesie, and Fennoscandia. The Biebrza group has experienced spatial and demographic expansion in the recent past. Average genetic differentiation among moose populations in Poland at mtDNA-cr was great and significant (ΦST = 0.407, p moose that colonized Poland from the east (Lithuania, Belarus, and Ukraine) and the north (former East Prussia). Among all the sequenced Y-chromosome markers, polymorphisms were found in the DBY14 marker in three populations only; four haplotypes were recorded in total. No significant differentiation was detected for this Y-linked marker among moose populations in Poland. Our mtDNA study revealed that a variety of different factors-bottleneck, the presence of relict, autochthonous populations, translocations, limited female dispersal, and the colonization from the east and north-are responsible for the observed complex pattern of population genetic structure after demographic and spatial expansion of moose in Poland.

  2. Subsurface Connections and Magma Mixing as revealed by Olivine- and Pyroxene-Hosted Melt Inclusions from Cerro Negro Volcano and the Las Pilas-El Hoyo Complex, Nicaragua.

    Science.gov (United States)

    Venugopal, S.; Moune, S.; Williams-Jones, G.

    2015-12-01

    Cerro Negro, the youngest volcano in the Central American Volcanic Belt, is a polygenetic cinder cone with relatively frequent explosive basaltic eruptions. Las Pilas, on the other hand, is a much larger and older complex with milder and less frequent eruptions. Based on historical data, these two closely spaced volcanoes have shown concurrent eruptive behavior, suggesting a subsurface connection. To further investigate this link, melt inclusions, which are blebs of melt trapped in growing crystals, were the obvious choice for optimal comparison of sources and determination of pre-eruptive volatile contents and magmatic conditions. Olivine-hosted inclusions were chosen for both volcanoes and pyroxene-hosted inclusions were also sampled from Las Pilas to represent the evolved melt. Major, volatile and trace elements reveal a distinct geochemical continuum with Cerro Negro defining the primitive end member and Las Pilas representing the evolved end member. Volatile contents are high for Cerro Negro (up to 1260 ppm CO2, 4.27 wt% H2O and 1700 ppm S) suggesting that volatile exsolution is likely the trigger for Cerro Negro's explosive eruptions. Las Pilas volatile contents are lower but consistent with degassing and evolutionary trends shown by major oxides. Trace element contents are rather unique and suggest Cerro Negro magmas fractionally crystallize while Las Pilas magmas are the products of mixing. Magmatic conditions were estimated with major and volatile contents: at least 2.4 kbar and 1170 °C for Cerro Negro melts and 1.3 kbar and 1130 °C for Las Pilas melts with an overall oxygen fugacity at the NNO buffer. In combination with available literature data, this study suggests an interconnected subsurface plumbing system and thus Cerro Negro should be considered as the newest vent within the Las Pilas-El Hoyo Complex.

  3. Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome.

    Directory of Open Access Journals (Sweden)

    Susan M Gribble

    Full Text Available Down syndrome (DS is caused by trisomy of chromosome 21 (Hsa21 and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1 presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2 overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

  4. Of mice and the 'Age of Discovery': the complex history of colonization of the Azorean archipelago by the house mouse (Mus musculus) as revealed by mitochondrial DNA variation.

    Science.gov (United States)

    Gabriel, S I; Mathias, M L; Searle, J B

    2015-01-01

    Humans have introduced many species onto remote oceanic islands. The house mouse (Mus musculus) is a human commensal and has consequently been transported to oceanic islands around the globe as an accidental stowaway. The history of these introductions can tell us not only about the mice themselves but also about the people that transported them. Following a phylogeographic approach, we used mitochondrial D-loop sequence variation (within an 849- to 864-bp fragment) to study house mouse colonization of the Azores. A total of 239 sequences were obtained from all nine islands, and interpretation was helped by previously published Iberian sequences and 66 newly generated Spanish sequences. A Bayesian analysis revealed presence in the Azores of most of the D-loop clades previously described in the domesticus subspecies of the house mouse, suggesting a complex colonization history of the archipelago as a whole from multiple geographical origins, but much less heterogeneity (often single colonization?) within islands. The expected historical link with mainland Portugal was reflected in the pattern of D-loop variation of some of the islands but not all. A more unexpected association with a distant North European source area was also detected in three islands, possibly reflecting human contact with the Azores prior to the 15th century discovery by Portuguese mariners. Widening the scope to colonization of the Macaronesian islands as a whole, human linkages between the Azores, Madeira, the Canaries, Portugal and Spain were revealed through the sharing of mouse sequences between these areas. From these and other data, we suggest mouse studies may help resolve historical uncertainties relating to the 'Age of Discovery'.

  5. Interaction of Di-2-pyridylketone 2-pyridine Carboxylic Acid Hydrazone and Its Copper Complex with BSA: Effect on Antitumor Activity as Revealed by Spectroscopic Studies.

    Science.gov (United States)

    Li, Cuiping; Huang, Tengfei; Fu, Yun; Liu, Youxun; Zhou, Sufeng; Qi, Zhangyang; Li, Changzheng

    2016-04-28

    The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity.

  6. Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development.

    Directory of Open Access Journals (Sweden)

    Nico Posnien

    2011-12-01

    Full Text Available Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.

  7. β-Lactoglobulin detected in human milk forms noncovalent complexes with maltooligosaccharides as revealed by chip-nanoelectrospray high-resolution tandem mass spectrometry.

    Science.gov (United States)

    Capitan, Florina; Robu, Adrian C; Schiopu, Catalin; Ilie, Constantin; Chait, Brian T; Przybylski, Michael; Zamfir, Alina D

    2015-11-01

    Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. β-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research. In this work, an innovative methodology encompassing microfluidics based on fully automated chip-nanoelectrospray ionization (nanoESI), coupled with high-resolution mass spectrometry (MS) on a quadrupole time-of-flight (QTOF MS) instrument was developed. This platform was employed for the assessment of the noncovalent interactions between maltohexaose (Glc6) and β-lactoglobulin extracted from human milk upon deliberate intake of cow's milk. The experiments were carried out in (+) ESI mode, using ammonium acetate (pH 6.0) as the buffer and also in pure water. In both cases, the MS analysis revealed the formation of BLG-Glc6 complex, which was characterized by top-down fragmentation in tandem MS (MS/MS) using collision-induced dissociation (CID). Our findings have a significant biomedical impact, indicating that Glc6 binds BLG under conditions mimicking the in vivo environment and therefore might represent a ligand, able to reduce its allergenicity.

  8. Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations

    Directory of Open Access Journals (Sweden)

    Anna Dowierciał

    2014-01-01

    Full Text Available The crystal structure of mouse thymidylate synthase (mTS in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB and thus supporting tighter binding of ligands, and the other being more open (dimer CD and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.

  9. Crystal structure of mouse thymidylate synthase in tertiary complex with dUMP and raltitrexed reveals N-terminus architecture and two different active site conformations.

    Science.gov (United States)

    Dowierciał, Anna; Wilk, Piotr; Rypniewski, Wojciech; Rode, Wojciech; Jarmuła, Adam

    2014-01-01

    The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.

  10. Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements.

    Science.gov (United States)

    Lundgren, Stina; Andersen, Birgit; Piskur, Jure; Dobritzsch, Doreen

    2007-12-07

    Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

  11. What do fault patterns reveal about the latest phase of extension within the Northern Snake Range metamorphic core complex, Nevada, USA?

    Science.gov (United States)

    Ismat, Zeshan; Riley, Paul; Lerback, Jory

    2016-08-01

    The Northern Snake Range is a classic example of a metamorphic core complex, Basin-and-Range province, United States. It is composed of a plastically deformed footwall and a brittlely deformed hanging wall, separated by the Northern Snake Range low-angle detachment (NSRD). Brittle deformation, however, is not confined to the hanging wall. This paper focuses on exposures in Cove Canyon, located on the SE flank of the Northern Snake Range, where penetrative, homogeneous faults are well exposed throughout the hanging wall, footwall and NSRD, and overprint early plastic deformation. These late-stage fault sets assisted Eocene-Miocene extension. Detailed analysis of the faults reveals the following: (1) The shortening direction defined by faults is similar to the shortening direction defined by the stretching lineation in the footwall mylonites, indicating that the extensional kinematic history remained unchanged as the rocks were uplifted into the elastico-frictional regime. (2) After ∼17 Ma, extension may have continued entirely within elastic-frictional regime via cataclastic flow. (3) This latest deformation phase may have been accommodated by a single, continuous event. (3) Faults within NSRD boudins indicate that deformation within the detachment zone was non-coaxial during the latest phase of extension.

  12. Time-dependent X-ray diffraction studies on urea/hen egg white lysozyme complexes reveal structural changes that indicate onset of denaturation

    Science.gov (United States)

    Raskar, Tushar; Khavnekar, Sagar; Hosur, Madhusoodan

    2016-01-01

    Temporal binding of urea to lysozyme was examined using X-ray diffraction of single crystals of urea/lysozyme complexes prepared by soaking native lysozyme crystals in solutions containing 9 M urea. Four different soak times of 2, 4, 7 and 10 hours were used. The five crystal structures (including the native lysozyme), refined to 1.6 Å resolution, reveal that as the soaking time increased, more and more first-shell water molecules are replaced by urea. The number of hydrogen bonds between urea and the protein is similar to that between protein and water molecules replaced by urea. However, the number of van der Waals contacts to protein from urea is almost double that between the protein and the replaced water. The hydrogen bonding and van der Waals interactions are initially greater with the backbone and later with side chains of charged residues. Urea altered the water-water hydrogen bond network both by replacing water solvating hydrophobic residues and by shortening the first-shell intra-water hydrogen bonds by 0.2 Å. These interaction data suggest that urea uses both ‘direct’ and ‘indirect’ mechanisms to unfold lysozyme. Specific structural changes constitute the first steps in lysozyme unfolding by urea. PMID:27573790

  13. Effects on hippocampus of lifelong absence of glucocorticoids in the pro-opiomelanocortin null mutant mouse reveal complex relationship between glucocorticoids and hippocampal structure and function.

    Science.gov (United States)

    Ostwald, Dirk; Karpac, Jason; Hochgeschwender, Ute

    2006-01-01

    In humans changes in serum cortisol levels have been observed with aging, stress, and with affective disorders such as major depression and post-traumatic stress disorder. Corticosteroids are known to influence hippocampal structure and function; specifically, plasma corticosteroid levels have been inversely correlated with hippocampal cell proliferation, cell death, and impaired memory function. The relationship between corticosteroids and structure and function of the hippocampus has been studied in experimental systems in adult animals by increasing or decreasing corticosterone levels through pharmacological supplementation and through surgical removal of the adrenal gland. Here, we utilized the genetically engineered pro-opiomelanocortin (POMC) null mutant mouse, which because of the lack of all POMC peptides has no corticosterone from birth throughout life. The effect of this lifelong absence of corticosterone on the dentate gyrus of the hippocampus is a decrease in granule cell density, which correlated with a decrease in cell proliferation but not an increase in cell degeneration. Fine morphology of granule cells was unaltered. Analyses of gene expression revealed no changes in POMC null mutant vs wild-type hippocampus with respect to levels of expression of corticoid receptor genes or genes known to be regulated by corticosterone. Spatial learning as tested by the Morris water maze was not altered in the POMC null mutant mouse. Taken together with findings from other studies of the effects of altered levels of corticosteroids on the hippocampus, our results argue for a complex homeostasis in which disturbances of any one factor can offset the system in varying ways.

  14. Copper(II)-bis-histidine coordination structure in a fibrillar amyloid β-peptide fragment and model complexes revealed by electron spin echo envelope modulation spectroscopy.

    Science.gov (United States)

    Hernández-Guzmán, Jessica; Sun, Li; Mehta, Anil K; Dong, Jijun; Lynn, David G; Warncke, Kurt

    2013-09-23

    Truncated and mutated amyloid-β (Aβ) peptides are models for systematic study-in homogeneous preparations-of the molecular origins of metal ion effects on Aβ aggregation rates, types of aggregate structures formed, and cytotoxicity. The 3D geometry of bis-histidine imidazole coordination of Cu(II) in fibrils of the nonapetide acetyl-Aβ(13-21)H14A has been determined by powder (14) N electron spin echo envelope modulation (ESEEM) spectroscopy. The method of simulation of the anisotropic combination modulation is described and benchmarked for a Cu(II) -bis-cis-imidazole complex of known structure. The revealed bis-cis coordination mode, and the mutual orientation of the imidazole rings, for Cu(II) in Ac-Aβ(13-21)H14A fibrils are consistent with the proposed β-sheet structural model and pairwise peptide interaction with Cu(II) , with an alternating [-metal-vacancy-]n pattern, along the N-terminal edge. Metal coordination does not significantly distort the intra-β-strand peptide interactions, which provides a possible explanation for the acceleration of Ac-Aβ(13-21)H14A fibrillization by Cu(II) , through stabilization of the associated state and low-reorganization integration of β-strand peptide pair precursors.

  15. Ancient DNA of the extinct lava shearwater (Puffinus olsoni from the Canary Islands reveals incipient differentiation within the P. puffinus complex.

    Directory of Open Access Journals (Sweden)

    Oscar Ramirez

    Full Text Available BACKGROUND: The loss of species during the Holocene was, dramatically more important on islands than on continents. Seabirds from islands are very vulnerable to human-induced alterations such as habitat destruction, hunting and exotic predators. For example, in the genus Puffinus (family Procellariidae the extinction of at least five species has been recorded during the Holocene, two of them coming from the Canary Islands. METHODOLOGY/PRINCIPAL FINDINGS: We used bones of the two extinct Canary shearwaters (P. olsoni and P. holeae to obtain genetic data, for use in providing insights into the differentiation process within the genus Puffinus. Although mitochondrial DNA (mtDNA cytochrome b sequences were successfully retrieved from four Holocene specimens of the extinct Lava shearwater (P. olsoni from Fuerteventura (Canary Islands, the P. holeae specimens yielded no DNA. Only one haplotype was detected in P. olsoni, suggesting a low genetic diversity within this species. CONCLUSIONS: The phylogenetic analyses based on the DNA data reveal that: (i the "Puffinus puffinus complex", an assemblage of species defined using osteological characteristics (P. puffinus, P. olsoni, P. mauretanicus, P. yelkouan and probably P. holeae, shows unresolved phylogenetic relationships; (ii despite the differences in body size and proportions, P. olsoni and the extant P. puffinus are sister species. Several hypotheses can be considered to explain the incipient differentiation between P. olsoni and P. puffinus.

  16. The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

    Science.gov (United States)

    Bao, Rui; Zhou, Cong-Zhao; Jiang, Chunhui; Lin, Sheng-Xiang; Chi, Cheng-Wu; Chen, Yuxing

    2009-09-25

    The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

  17. Mating type locus of Chinese black truffles reveals heterothallism and the presence of cryptic species within the T. indicum species complex.

    Directory of Open Access Journals (Sweden)

    Beatrice Belfiori

    Full Text Available Tuber spp. are filamentous ascomycetes which establish symbiosis with the roots of trees and shrub species. By virtue of this symbiosis they produce hypogeous ascocarps, known as truffles. Filamentous ascomycetes can reproduce by homothallism or heterothallism depending on the structure and organization of their mating type locus. The first mating type locus in a truffle species has been recently characterized in Tuber melanosporum and it has been shown that this fungus, endemic in Europe, is heterothallic. The availability of sequence information for T. melanosporum mating type genes is seminal to cloning their orthologs from other Tuber species and assessing their reproductive mode. Here we report on the organization of the mating type region in T. indicum, the black truffle species present in Asia, which is the closest relative to T. melanosporum and is characterized by an high level of morphological and genetic variability. The present study shows that T. indicum is also heterothallic. Examination of Asiatic black truffles belonging to different genetic classes, sorted according to the sequence polymorphism of the internal transcribed spacer rDNA region, has revealed sequence variations and rearrangements in both coding and non-coding regions of the mating type locus, to suggest the existence of cryptic species within the T. indicum complex. The presence of transposable elements within or linked to the mating type region suggests a role of these elements in generating the genotypic diversity present among T. indicum strains. Overall, comparative analyses of the mating type locus have thus allowed us to tackle taxonomical and phylogenetic issues within black truffles and make inferences about the evolution of T. melanosporum-T. indicum lineage. Our results are not only of fundamental but also of applied relevance as T. indicum produces edible fruit bodies that are imported also into Europe and thus may represent a biological threat for T

  18. Study of Mycobacterium tuberculosis complex genotypic diversity in Malaysia reveals a predominance of ancestral East-African-Indian lineage with a Malaysia-specific signature.

    Science.gov (United States)

    Ismail, Fazli; Couvin, David; Farakhin, Izzah; Abdul Rahman, Zaidah; Rastogi, Nalin; Suraiya, Siti

    2014-01-01

    Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs. This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database. Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (pMalaysia, and its probable ongoing evolution with locally evolved strains sharing a specific signature characterized by absence of spacers 37, 38, and 40. Pending complementary genotyping confirmation, we propose that SIT745/EAI-SOM is tentatively reclassified as SIT745/EAI-MYS.

  19. A Multiple Interaction Analysis Reveals ADRB3 as a Potential Candidate for Gallbladder Cancer Predisposition via a Complex Interaction with Other Candidate Gene Variations

    Directory of Open Access Journals (Sweden)

    Rajani Rai

    2015-11-01

    Full Text Available Gallbladder cancer is the most common and a highly aggressive biliary tract malignancy with a dismal outcome. The pathogenesis of the disease is multifactorial, comprising the combined effect of multiple genetic variations of mild consequence along with numerous dietary and environmental risk factors. Previously, we demonstrated the association of several candidate gene variations with GBC risk. In this study, we aimed to identify the combination of gene variants and their possible interactions contributing towards genetic susceptibility of GBC. Here, we performed Multifactor-Dimensionality Reduction (MDR and Classification and Regression Tree Analysis (CRT to investigate the gene–gene interactions and the combined effect of 14 SNPs in nine genes (DR4 (rs20576, rs6557634; FAS (rs2234767; FASL (rs763110; DCC (rs2229080, rs4078288, rs7504990, rs714; PSCA (rs2294008, rs2978974; ADRA2A (rs1801253; ADRB1 (rs1800544; ADRB3 (rs4994; CYP17 (rs2486758 involved in various signaling pathways. Genotyping was accomplished by PCR-RFLP or Taqman allelic discrimination assays. SPSS software version 16.0 and MDR software version 2.0 were used for all the statistical analysis. Single locus investigation demonstrated significant association of DR4 (rs20576, rs6557634, DCC (rs714, rs2229080, rs4078288 and ADRB3 (rs4994 polymorphisms with GBC risk. MDR analysis revealed ADRB3 (rs4994 to be crucial candidate in GBC susceptibility that may act either alone (p < 0.0001, CVC = 10/10 or in combination with DCC (rs714 and rs2229080, p < 0.0001, CVC = 9/10. Our CRT results are in agreement with the above findings. Further, in-silico results of studied SNPs advocated their role in splicing, transcriptional and/or protein coding regulation. Overall, our result suggested complex interactions amongst the studied SNPs and ADRB3 rs4994 as candidate influencing GBC susceptibility.

  20. Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design.

    Science.gov (United States)

    Mabanglo, Mark F; Hast, Michael A; Lubock, Nathan B; Hellinga, Homme W; Beese, Lorena S

    2014-03-01

    Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate-binding step is accompanied by motions of a loop in the catalytic site. Re-examination of other FTase structures showed that this motion is conserved. The substrate- and product-binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α-helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady-state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti-fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.

  1. Metabolite profiles reveal energy failure and impaired beta-oxidation in liver of mice with complex III deficiency due to a BCS1L mutation.

    Directory of Open Access Journals (Sweden)

    Heike Kotarsky

    Full Text Available BACKGROUND & AIMS: Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. METHODS: With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H(2O(2 production and expression of antioxidants. RESULTS: Homozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. CONCLUSIONS: The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.

  2. Mapping Breakpoints of Complex Chromosome Rearrangements Involving a Partial Trisomy 15q23.1-q26.2 Revealed by Next Generation Sequencing and Conventional Techniques.

    Directory of Open Access Journals (Sweden)

    Qiong Pan

    Full Text Available Complex chromosome rearrangements (CCRs, which are rather rare in the whole population, may be associated with aberrant phenotypes. Next-generation sequencing (NGS and conventional techniques, could be used to reveal specific CCRs for better genetic counseling. We report the CCRs of a girl and her mother, which were identified using a combination of NGS and conventional techniques including G-banding, fluorescence in situ hybridization (FISH and PCR. The girl demonstrated CCRs involving chromosomes 3 and 8, while the CCRs of her mother involved chromosomes 3, 5, 8, 11 and 15. HumanCytoSNP-12 Chip analysis identified a 35.4 Mb duplication on chromosome 15q21.3-q26.2 in the proband and a 1.6 Mb microdeletion at chromosome 15q21.3 in her mother. The proband inherited the rearranged chromosomes 3 and 8 from her mother, and the duplicated region on chromosome 15 of the proband was inherited from the mother. Approximately one hundred genes were identified in the 15q21.3-q26.2 duplicated region of the proband. In particular, TPM1, SMAD6, SMAD3, and HCN4 may be associated with her heart defects, and HEXA, KIF7, and IDH2 are responsible for her developmental and mental retardation. In addition, we suggest that a microdeletion on the 15q21.3 region of the mother, which involved TCF2, TCF12, ADMA10 and AQP9, might be associated with mental retardation. We delineate the precise structures of the derivative chromosomes, chromosome duplication origin and possible molecular mechanisms for aberrant phenotypes by combining NGS data with conventional techniques.

  3. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Directory of Open Access Journals (Sweden)

    Hongtao Hu

    Full Text Available MicroRNAs (miRNAs and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs are two distinct subfamilies of small RNAs (sRNAs that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs are processed from longer RNA precursors by DICER-LIKE proteins (DCLs. Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs and 108 novel lineage-specific miRNAs (ls-miRNAs. Along with miRNAs, 2,033 miRNA variants (isomiRNAs were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers

  4. Study of Mycobacterium tuberculosis complex genotypic diversity in Malaysia reveals a predominance of ancestral East-African-Indian lineage with a Malaysia-specific signature.

    Directory of Open Access Journals (Sweden)

    Fazli Ismail

    Full Text Available Tuberculosis (TB still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC isolates causing the disease is important to determine the effectiveness of the control and surveillance programs.This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database.Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe.Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT designation in the database (the latter included 6 newly created SITs. The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01. However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades.The ancestral East-African-Indian (EAI lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for Malaysia, and its probable ongoing evolution

  5. Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer

    Directory of Open Access Journals (Sweden)

    Kenter Gemma G

    2007-02-01

    Full Text Available Abstract Background Cervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH and microsatellite marker analysis for the detection of loss of heterozygosity (LOH. Currently, high throughput methods such as array comparative genomic hybridization (array CGH, single nucleotide polymorphism array (SNP array and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression. Results Genome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH. Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR. Conclusion This study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene

  6. The venom-gland transcriptome of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the intragenomic evolution of venoms.

    Science.gov (United States)

    Margres, Mark J; Aronow, Karalyn; Loyacano, Jacob; Rokyta, Darin R

    2013-08-02

    Snake venom is shaped by the ecology and evolution of venomous species, and signals of positive selection in toxins have been consistently documented, reflecting the role of venoms as an ecologically critical phenotype. New World coral snakes (Elapidae) are represented by three genera and over 120 species and subspecies that are capable of causing significant human morbidity and mortality, yet coral-snake venom composition is poorly understood in comparison to that of Old World elapids. High-throughput sequencing is capable of identifying thousands of loci, while providing characterizations of expression patterns and the molecular evolutionary forces acting within the venom gland. We describe the de novo assembly and analysis of the venom-gland transcriptome of the eastern coral snake (Micrurus fulvius). We identified 1,950 nontoxin transcripts and 116 toxin transcripts. These transcripts accounted for 57.1% of the total reads, with toxins accounting for 45.8% of the total reads. Phospholipases A(2) and three-finger toxins dominated expression, accounting for 86.0% of the toxin reads. A total of 15 toxin families were identified, revealing venom complexity previously unknown from New World coral snakes. Toxins exhibited high levels of heterozygosity relative to nontoxins, and overdominance may favor gene duplication leading to the fixation of advantageous alleles. Phospholipase A(2) expression was uniformly distributed throughout the class while three-finger toxin expression was dominated by a handful of transcripts, and phylogenetic analyses indicate that toxin divergence may have occurred following speciation. Positive selection was detected in three of the four most diverse toxin classes, suggesting that venom diversification is driven by recurrent directional selection. We describe the most complete characterization of an elapid venom gland to date. Toxin gene duplication may be driven by heterozygote advantage, as the frequency of polymorphic toxin loci was

  7. Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication

    Directory of Open Access Journals (Sweden)

    Bryder David

    2010-02-01

    Full Text Available Abstract Background The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow. Results In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development, OP-9 cells (strongly supportive of B cell development, pro-B and pre-B cells as well as mature peripheral B-lineage cells, we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further, the analysis suggested that there existed possibilities for progenitor B cells to send signals to

  8. Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2

    DEFF Research Database (Denmark)

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle;

    2010-01-01

    We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely relate...

  9. Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria.

    Science.gov (United States)

    Huergo, Luciano F; Merrick, Mike; Pedrosa, Fábio O; Chubatsu, Leda S; Araujo, Luíza M; Souza, Emanuel M

    2007-12-01

    Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.

  10. Mutational Analysis of the QRRQ Motif in the Yeast Hig1-type 2 Protein, Rcf1, Reveals a Regulatory Role for the Cytochrome c Oxidase Complex.

    Science.gov (United States)

    Garlich, Joshua; Strecker, Valentina; Wittig, Ilka; Stuart, Rosemary A

    2017-02-06

    The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system in particular the cytochrome bc1 (complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins (AAC). Rcf1 plays a role in the assembly and modulation of the activity of complex IV, however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1-type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)-X3-(R/H)-X-R-X3-Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and an alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and in doing so regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and possibly other mitochondrial proteins, such as AAC.

  11. In vitro targeting reveals intrinsic histone tail specificity of the Sin3/histone deacetylase and N-CoR/SMRT corepressor complexes.

    NARCIS (Netherlands)

    Vermeulen, M.; Carrozza, M.J.; Lasonder, E.; Workman, J.L.; Logie, C.; Stunnenberg, H.G.

    2004-01-01

    The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of

  12. RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions

    DEFF Research Database (Denmark)

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, onl...

  13. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    Energy Technology Data Exchange (ETDEWEB)

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  14. Structures of the HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of the AIM2 Inflammasome and IFI16 Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Tengchuan; Perry, Andrew; Jiang, Jiansheng; Smith, Patrick; Curry, James A.; Unterholzner, Leonie; Jiang, Zhaozhao; Horvath, Gabor; Rathinam, Vijay A.; Johnstone, Ricky W.; Hornung, Veit; Latz, Eicke; Bowie, Andrew G.; Fitzgerald, Katherine A.; Xiao, T. Sam (UMASS, MED); (Bonn); (Trinity); (PMCI-A); (NIH)

    2012-05-21

    Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.

  15. Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.

    Directory of Open Access Journals (Sweden)

    Anke Vermehren-Schmaedick

    Full Text Available Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes. Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are

  16. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

    DEFF Research Database (Denmark)

    Vorup-Jensen, T; Petersen, Steen Vang; Hansen, A G;

    2000-01-01

    Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine...

  17. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    Science.gov (United States)

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit

    DEFF Research Database (Denmark)

    Paës, Gabriel; Skov, Lars K; O'Donohue, Michael J

    2008-01-01

    The crystal structure of the family GH-51 alpha- l-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural ...

  19. Crystal Structure of the VapBC Toxin–Antitoxin Complex from Shigella flexneri Reveals a Hetero-Octameric DNA-Binding Assembly

    DEFF Research Database (Denmark)

    Dienemann, Christian; Bøggild, Andreas; Winther, Kristoffer S.

    2011-01-01

    the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total...

  20. Neuroarchitecture of the central complex in the brain of the locust Schistocerca gregaria and S. americana as revealed by serotonin immunocytochemistry.

    Science.gov (United States)

    Homberg, U

    1991-01-08

    The central complex is a prominent structure in the insect brain, yet its anatomical organization and functional role is still poorly understood. To facilitate investigations on the physiology of the central complex, this study describes its anatomical organization in the brain of locusts (Schistocerca gregaria and Schistocerca americana) based on an investigation of serotonin immunocytochemistry. Most subdivisions of the central complex including the protocerebral bridge, several layers in the upper division of the central body, and the noduli of the central body are innervated by serotonin-immunoreactive neurons, while the lower division of the central body does not exhibit serotonin-like immunoreactivity. Several types of serotonin-immunoreactive neurons can be distinguished. A system of about 60 columnar neurons innervates the protocerebral bridge, layer III of the upper division of the central body, and the noduli. A group of 15-20 bilateral pairs of serotonin-immunoreactive neurons connects the posterior optic tubercles with the protocerebral bridge. About ten pairs of neurons with somata in the inferior protocerebrum innervate layer Ia of the upper division of the central body. In addition, large-field neurons arborize in layers Ia and Ib of the upper division of the central body and in the lateral accessory lobes. The detailed mapping of serotonin immunoreactivity provides further insight into the anatomical organization of the central complex and suggests that serotonin is a major neuroactive substance within this brain structure.

  1. Resonant X-ray emission spectroscopy reveals d-d ligand-field states involved in the self-assembly of a square-planar platinum complex.

    Science.gov (United States)

    Garino, Claudio; Gallo, Erik; Smolentsev, Nikolay; Glatzel, Pieter; Gobetto, Roberto; Lamberti, Carlo; Sadler, Peter J; Salassa, Luca

    2012-11-28

    Resonant X-ray Emission Spectroscopy (RXES) is used to characterize the ligand field states of the prototypic self-assembled square-planar complex, [Pt(tpy)Cl]Cl (tpy=2,2':6',2''-terpyridine), and determine the effect of weak metal-metal and π-π interactions on their energy.

  2. Sequential karyotyping in Burkitt lymphoma reveals a linear clonal evolution with increase in karyotype complexity and a high frequency of recurrent secondary aberrations

    NARCIS (Netherlands)

    Aukema, Sietse M.; Theil, Laura; Rohde, Marius; Bauer, Benedikt; Bradtke, Jutta; Burkhardt, Birgit; Bonn, Bettina R.; Claviez, Alexander; Gattenloehner, Stefan; Makarova, Olga; Nagel, Inga; Oschlies, Ilske; Pott, Christiane; Szczepanowski, Monika; Traulsen, Arne; Kluin, Philip M.; Klapper, Wolfram; Siebert, Reiner; Penas, Eva M. Murga

    2015-01-01

    Typical Burkitt lymphoma is characterized by an IG-MYC translocation and overall low genomic complexity. Clinically, Burkitt lymphoma has a favourable prognosis with very few relapses. However, the few patients experiencing disease progression and/or relapse have a dismal outcome. Here we report cyt

  3. Complexity in Climatic Controls on Plant Species Distribution: Satellite Data Reveal Unique Climate for Giant Sequoia in the California Sierra Nevada

    Science.gov (United States)

    Waller, Eric Kindseth

    governing the distribution? Detailed aspects of the local climate warranted more investigation. Chapter 4 investigates the climate associated with the frequent cloud formation over the western slopes of the southern Sierra Nevada: the "sequoia belt". This region is climatically distinct in a number of ways, all of which could be factors in influencing the distribution of giant sequoia and other species. Satellite and micrometeorological flux tower data reveal characteristics of the sequoia belt that were not evident with surface climate measurements and maps derived from them. Results have implications for species distributions everywhere, but especially in rugged mountains, where climates are complex and poorly mapped. Chapter 5 summarizes some of the main conclusions from the work and suggests directions for related future research. (Abstract shortened by UMI.).

  4. Tapping to a slow tempo in the presence of simple and complex meters reveals experience-specific biases for processing music.

    Directory of Open Access Journals (Sweden)

    Sangeeta Ullal-Gupta

    Full Text Available Musical meters vary considerably across cultures, yet relatively little is known about how culture-specific experience influences metrical processing. In Experiment 1, we compared American and Indian listeners' synchronous tapping to slow sequences. Inter-tone intervals contained silence or to-be-ignored rhythms that were designed to induce a simple meter (familiar to Americans and Indians or a complex meter (familiar only to Indians. A subset of trials contained an abrupt switch from one rhythm to another to assess the disruptive effects of contradicting the initially implied meter. In the unfilled condition, both groups tapped earlier than the target and showed large tap-tone asynchronies (measured in relative phase. When inter-tone intervals were filled with simple-meter rhythms, American listeners tapped later than targets, but their asynchronies were smaller and declined more rapidly. Likewise, asynchronies rose sharply following a switch away from simple-meter but not from complex-meter rhythm. By contrast, Indian listeners performed similarly across all rhythm types, with asynchronies rapidly declining over the course of complex- and simple-meter trials. For these listeners, a switch from either simple or complex meter increased asynchronies. Experiment 2 tested American listeners but doubled the duration of the synchronization phase prior to (and after the switch. Here, compared with simple meters, complex-meter rhythms elicited larger asynchronies that declined at a slower rate, however, asynchronies increased after the switch for all conditions. Our results provide evidence that ease of meter processing depends to a great extent on the amount of experience with specific meters.

  5. Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis*

    OpenAIRE

    Chuankhayan, Phimonphan; Hsieh, Chih-Yu; Huang, Yen-Chieh; Hsieh, Yi-You; Guan, Hong-Hsiang; Hsieh, Yin-Cheng; Tien, Yueh-Chu; Chen, Chung-De; Chiang, Chien-Min; Chen, Chun-Jung

    2010-01-01

    Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and ...

  6. Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design

    OpenAIRE

    Mabanglo, Mark F.; Hast, Michael A.; Lubock, Nathan B; Hellinga, Homme W.; Beese, Lorena S.

    2014-01-01

    Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide ...

  7. The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes

    Directory of Open Access Journals (Sweden)

    Hélène eHardré

    2014-05-01

    Full Text Available The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM, based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM might need the integrity of a trans-envelope (IEM-OEM protein complex (e.g. division ring-forming components or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.

  8. Negative Ion Photoelectron Spectroscopy Reveals Remarkable Noninnocence of Ligands in Nickel Bis(dithiolene) Complexes [Ni(dddt)2](-) and [Ni(edo)2](.).

    Science.gov (United States)

    Liu, Xing; Hou, Gao-Lei; Wang, Xuefeng; Wang, Xue-Bin

    2016-05-12

    [Ni(dddt)2](-) (dddt = 5,6-dihydro-1,4-dithiine-2,3-dithiolate) and [Ni(edo)2](-) (edo = 5,6-dihydro-1,4-dioxine-2,3-dithiolate) are two donor-type nickel bis(dithiolene) complexes, with the tendency of donating low binding energy electrons. These two structurally similar complexes differ only with respect to the outer atoms in the ligand framework where the former has four S atoms while the latter has four O atoms. Herein, we report a negative ion photoelectron spectroscopy (NIPES) study on these two complexes to probe the electronic structures of the anions and their corresponding neutrals. The NIPE spectra exhibit the adiabatic electron detachment energy (ADE) or, equivalently, the electron affinity (EA) of the neutral [Ni(L)2](0) to be relatively low for this type of complexes, 2.780 and 2.375 eV for L = dddt and edo, respectively. The 0.4 eV difference in ADEs shows a significant substitution effect for sulfur in dddt by oxygen in edo, i.e., noninnocence of the ligands, which has decreased the electronic stability of [Ni(edo)2](-) by lowering its electron binding energy by ∼0.4 eV. The observed substitution effect on gas-phase EA values correlates well with the measured redox potentials for [Ni(dddt)2](-/0) and [Ni(edo)2](-/0) in solutions. The singlet-triplet splitting (ΔEST) of [Ni(dddt)2](0) and [Ni(edo)2](0) is also determined from the spectra to be 0.57 and 0.53 eV, respectively. Accompanying DFT calculations and molecular orbital (MO) composition analyses show significant ligand contributions to the redox MOs and allow the components of the orbitals involved in each electronic transition and spectral assignments to be identified.

  9. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-09-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  10. Binding of the Respiratory Chain Inhibitor Antimycin to theMitochondrial bc1 Complex: A New Crystal Structure Reveals an AlteredIntramolecular Hydrogen-Bonding Pattern

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Cobessi, David; Tung, Eric Y.; Berry, Edward A.

    2005-05-10

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex.Structure-activity-relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28Angstrom resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cyt b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alpha-A helix.

  11. Structural analysis of the Sil1-Bip complex reveals the mechanism for Sil1 to function as a nucleotide-exchange factor

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Ming; Li, Jingzhi; Sha, Bingdong (UAB)

    2013-01-16

    Sil1 functions as a NEF (nucleotide-exchange factor) for the ER (endoplasmic reticulum) Hsp70 (heat-shock protein of 70 kDa) Bip in eukaryotic cells. Sil1 may catalyse the ADP release from Bip by interacting directly with the ATPase domain of Bip. In the present study we show the complex crystal structure of the yeast Bip and the NEF Sil1 at the resolution of 2.3 {angstrom} (1 {angstrom} = 0.1 nm). In the Sil1-Bip complex structure, the Sil1 molecule acts as a 'clamp' which binds lobe IIb of the Bip ATPase domain. The binding of Sil1 causes the rotation of lobe IIb {approx} 13.5{sup o} away from the ADP-binding pocket. The complex formation also induces lobe Ib to swing in the opposite direction by {approx} 3.7{sup o}. These conformational changes open up the nucleotide-binding pocket in the Bip ATPase domain and disrupt the hydrogen bonds between Bip and bound ADP, which may catalyse ADP release. Mutation of the Sil1 residues involved in binding the Bip ATPase domain compromise the binding affinity of Sil1 to Bip, and these Sil1 mutants also abolish the ability to stimulate the ATPase activity of Bip.

  12. A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues.

    Science.gov (United States)

    Joanne Wang, C; Li, Xiong; Lin, Benjamin; Shim, Sangwoo; Ming, Guo-Li; Levchenko, Andre

    2008-02-01

    Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.

  13. Complexity in Climatic Controls on Plant Species Distribution: Satellite Data Reveal Unique Climate for Giant Sequoia in the California Sierra Nevada

    OpenAIRE

    Waller, Eric Kindseth

    2014-01-01

    ABSTRACTComplexity in Climatic Controls on Plant Species Distribution: Satellite Data Reveal Unique Climate for Giant Sequoia in the California Sierra NevadabyEric Kindseth WallerDoctor of Philosophy in Environmental Science, Policy, and ManagementUniversity of California, BerkeleyProfessor Dennis D. Baldocchi, ChairA better understanding of the environmental controls on current plant species distribution is essential if the impacts of such diverse challenges as invasive species, changing fir...

  14. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

    KAUST Repository

    Kim, Jeong Joo

    2016-04-09

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

  15. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    Science.gov (United States)

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  16. Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Starbird, C.A.; Maklashina, Elena; Sharma, Pankaj; Qualls-Histed, Susan; Cecchini, Gary; Iverson, T.M. (VA); (UCSF); (Vanderbilt)

    2017-06-14

    The Escherichia coli Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the E. coli QFR FrdA subunit. Using a ΔsdhE E. coli strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdAE245 residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdAE245Q have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdAE245 variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdAE245 residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.

  17. Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain.

    Science.gov (United States)

    Sevcík, Jozef; Hostinová, Eva; Solovicová, Adriana; Gasperík, Juraj; Dauter, Zbigniew; Wilson, Keith S

    2006-05-01

    Most glucoamylases (alpha-1,4-D-glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 A and that of the enzyme-acarbose complex at 1.6 A resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo-cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the 'sugar tongs' surface binding site in the structure of barley alpha-amylase isozyme 1 complexed with a thiomalto-oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch-degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.

  18. LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1.

    Science.gov (United States)

    Pätsi, Jukka; Maliniemi, Pilvi; Pakanen, Salla; Hinttala, Reetta; Uusimaa, Johanna; Majamaa, Kari; Nyström, Thomas; Kervinen, Marko; Hassinen, Ilmo E

    2012-02-01

    Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.

  19. The origin of the unusual Qy red shift in LH1-RC complexes from purple bacteria Thermochromatium tepidum as revealed by Stark absorption spectroscopy.

    Science.gov (United States)

    Ma, Fei; Yu, Long-Jiang; Wang-Otomo, Zheng-Yu; van Grondelle, Rienk

    2015-12-01

    Native LH1-RC of photosynthetic purple bacteria Thermochromatium (Tch.) tepidum, B915, has an ultra-red BChl a Qy absorption. Two blue-shifted complexes obtained by chemical modification, B893 and B882, have increasing full widths at half maximum (FWHM) and decreasing transition dipole oscillator strength. 77K Stark absorption spectroscopy studies were employed for the three complexes, trying to understand the origin of the 915 nm absorption. We found that Tr(∆α) and |∆μ| of both Qy and carotenoid (Car) bands are larger than for other purple bacterial LH complexes reported previously. Moreover, the red shifts of the Qy bands are associated with (1) increasing Tr(∆α) and |∆μ| of the Qy band, (2) the red shift of the Car Stark signal and (3) the increasing |∆μ| of the Car band. Based on the results and the crystal structure, a combined effect of exciton-charge transfer (CT) states mixing, and inhomogeneous narrowing of the BChl a site energy is proposed to be the origin of the 915 nm absorption. CT-exciton state mixing has long been found to be the origin of strong Stark signal in LH1 and special pair, and the more extent of the mixing in Tch. tepidum LH1 is mainly the consequence of the shorter BChl-BChl distances. The less flexible protein structure results in a smaller site energy disorder (inhomogeneous narrowing), which was demonstrated to be able to influence |∆μ| and absorption.

  20. Molecular basis for shared cytokine recognition revealed in the structure of an unusually high affinity complex between IL-13 and IL-13Rα2

    OpenAIRE

    Lupardus, Patrick J.; Birnbaum, Michael E.; Garcia, K. Christopher

    2010-01-01

    Interleukin-13 is a cytokine important for development of T-helper cell type 2 (Th2) responses and plays a critical role in asthma and allergy. The IL-13 Receptor α2 (IL-13Rα2) is a receptor for IL-13 lacking canonical Jak/STAT signaling functions. Here we present the crystal structure along with a mutational and biophysical analysis of the IL-13/IL-13Rα2 complex. While retaining a similar mode of IL-13 binding to its related signaling receptor IL-13Rα1, IL-13Rα2 utilizes peripheral receptor ...

  1. 4-D MRI flow analysis in the course of interrupted aortic arch reveals complex morphology and quantifies amount of collateral blood flow

    Energy Technology Data Exchange (ETDEWEB)

    Hirtler, Daniel [University Hospital Freiburg, Department of Pediatric Cardiology and Congenital Heart Disease, Freiburg (Germany); Geiger, Julia; Jung, Bernd [University Hospital Freiburg, Department of Radiology, Medical Physics, Freiburg (Germany); Markl, Michael [Northwestern University, Departments of Radiology and Biomedical Engineering, Chicago, IL (United States); Arnold, Raoul [University Hospital Heidelberg, Department of Pediatric Cardiology and Congenital Heart Disease, Heidelberg (Germany)

    2013-08-15

    We present findings in a 17-year-old with interrupted aortic arch, in whom standard imaging techniques missed functional and morphological problems. Flow-sensitive four-dimensional magnetic resonance (4-D MR) enabled assessment of the complex anatomy and blood-flow characteristics in the entire aorta and direct quantification of blood flow in collateral vessels. Our findings highlight the entire morphological and functional problem of interrupted aortic arch and illustrate the potential of flow-sensitive 4-D MR for surgical planning in congenital heart disease. (orig.)

  2. Ear-body lift and a novel thrust generating mechanism revealed by the complex wake of brown long-eared bats (Plecotus auritus)

    DEFF Research Database (Denmark)

    Johansson, L Christoffer; Håkansson, Jonas; Jakobsen, Lasse;

    2016-01-01

    into the next wingbeat and rotates together with a newly formed tip vortex. Several smaller vortices, related to changes in circulation around the wing also spiral the tip vortex. Our results thus show a new level of complexity in bat wakes and suggest large eared bats are less aerodynamically limited than....... We also propose that the bats use a novel wing pitch mechanism at the end of the upstroke generating thrust at low speeds, which should provide effective pitch and yaw control. In addition, the wing tip vortices show a distinct spiraling pattern. The tip vortex of the previous wingbeat remains...... previous wake studies have suggested....

  3. Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd(2+)/Mg (2+)-con-T[K7gamma] complex.

    Science.gov (United States)

    Cnudde, Sara E; Prorok, Mary; Castellino, Francis J; Geiger, James H

    2010-06-01

    Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in gamma-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-D: -aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca(2+) and Mg(2+) can fulfill this role, Ca(2+) induces dimerization of con-G, whereas the Mg(2+)-complexed peptide remains monomeric. A variant of con-T, con-T[K7gamma] (gamma is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca(2+) ions and two Mg(2+) ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca(2+) for dimer formation, we report here the structure of the monomeric Cd(2+)/Mg(2+)-con-T[K7gamma] complex, and, by comparison with the previously published con-T[K7gamma]/Ca(2+) dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.

  4. Fingerprinting stress: stylolite and calcite twinning paleopiezometry reveal the complexity of stress distribution during the growth of the Monte Nero anticline (Apennines, Italy).

    Science.gov (United States)

    Beaudoin, Nicolas; Koehn, Daniel; Lacombe, Olivier; Lecouty, Alexandre; Billi, Andrea; Aharonov, Einat; Parlangeau, Camille

    2016-04-01

    This contribution presents for the first time how quantitative stress estimates can be derived by combining calcite twinning and stylolite roughness stress fingerprinting techniques in a structure part of a complex fold and thrust belts. We report a high-resolution deformation and stress history that was experienced by Meso-Cenozoic limestone strata in the overturned Monte Nero Anticline during its late Miocene-Pliocene growth in the Umbria-Marche Arcuate Ridge (northern Apennines, Italy). New methodological development enables an easier use for the inversion technique of sedimentary and tectonic stylolite roughness. A stylolite-fracture network developed during layer-parallel shortening (LPS), as well as syn- and post-folding. Stress fingerprinting shows how stress builds up in the sedimentary strata during LPS with variations of differential stress before folding around a value of 50 MPa. The stress regime oscillated between strike-slip and compressional during LPS and became transiently extensional in limbs of developing fold due to a coeval increase of vertical stress related to local burial and decrease of maximum horizontal stress related to hinge development, before ultimately becoming strike-slip again during late stage fold tightening. Our case study shows that stress fingerprinting is possible and that this novel method can be used to unravel complex temporal relationships that relate to local variations within evolving regional orogenic stresses. Beyond regional implication, this study validates our approach as a new exciting toolbox to high-resolution stress fingerprinting in basins and orogens.

  5. The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins.

    Science.gov (United States)

    Yatime, Laure; Betzer, Cristine; Jensen, Rasmus Kjeldsen; Mortensen, Sofia; Jensen, Poul Henning; Andersen, Gregers Rom

    2016-12-06

    S100 proteins are calcium-dependent regulators of homeostatic processes. Upon cellular response to stress, and notably during tumorigenesis, they relocalize to the extracellular environment where they induce pro-inflammatory signals by activating the receptor for advanced glycation end products (RAGE), thereby facilitating tumor growth and metastasis. Despite its importance in sustaining inflammation, the structural basis for RAGE-S100 crosstalk is still unknown. Here we report two crystal structures of the RAGE:S100A6 complex encompassing a full-length RAGE ectodomain. The structures, in combination with a comprehensive interaction analysis, suggest that the primary S100A6 binding site is formed by the RAGE C1 domain. Complex formation with S100A6 induces a unique dimeric conformation of RAGE that appears suited for signal transduction and intracellular effector recruitment. Intriguingly, S100A6 adopts a dimeric conformation radically different from all known S100 dimers. We discuss the physiological relevance of this non-canonical homodimeric form in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Single-Molecule Spectroscopy Reveals that Individual Low-Light LH2 Complexes from Rhodopseudomonas palustris 2.1.6. Have a Heterogeneous Polypeptide Composition

    Science.gov (United States)

    Brotosudarmo, Tatas H.P.; Kunz, Ralf; Böhm, Paul; Gardiner, Alastair T.; Moulisová, Vladimíra; Cogdell, Richard J.; Köhler, Jürgen

    2009-01-01

    Abstract Rhodopseudomonas palustris belongs to the group of purple bacteria that have the ability to produce LH2 complexes with unusual absorption spectra when they are grown at low-light intensity. This ability is often related to the presence of multiple genes encoding the antenna apoproteins. Here we report, for the first time to our knowledge, direct evidence that individual low-light LH2 complexes have a heterogeneous αβ-apoprotein composition that modulates the site energies of Bchl a molecules, producing absorption bands at 800, 820, and 850 nm. The arrangement of the Bchl a molecules in the “tightly coupled ring” can be modeled by nine αβ-Bchls dimers, such that the Bchls bound to six αβ-pairs have B820-like site energies and the remaining Bchl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these six αβ-pairs with B820 Bchl a molecules are distributed such that the symmetry of the assembly is reduced to C3. It is also clear from the measured single-molecule spectra that the energies of the electronically excited states in the mixed B820/850 ring are mainly influenced by diagonal disorder. PMID:19720038

  7. Comparative Proteomics Reveals Strain-Specific β-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex.

    Directory of Open Access Journals (Sweden)

    Siyuan Ding

    2016-10-01

    Full Text Available Rotaviruses (RVs are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1, a well-characterized interferon (IFN antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL complexes were identified. Importantly, inhibition of cullin-3 (Cul3 or RING-box protein 1 (Rbx1, by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.

  8. Analysis of meiosis in SUN1 deficient mice reveals a distinct role of SUN2 in mammalian meiotic LINC complex formation and function.

    Directory of Open Access Journals (Sweden)

    Jana Link

    2014-02-01

    Full Text Available LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84 and KASH (Klarsicht/ANC-1/Syne/homology domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1(-/- meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1(-/- mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.

  9. A novel cluster of Mycobacterium abscessus complex revealed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Suzuki, Hiromichi; Yoshida, Shiomi; Yoshida, Atsushi; Okuzumi, Katsuko; Fukusima, Atsuhito; Hishinuma, Akira

    2015-12-01

    Mycobacterium abscessus complex is a rapidly growing mycobacterium consisting of 3 subspecies, M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. However, rapid and accurate species identification is difficult. We first evaluated a suitable protocol of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for distinguishing these subspecies. Then, we studied spectral signals by MALDI-TOF MS in 59 M. abscessus, 42 M. massiliense, and 2 M. bolletii. Among several specific spectral signals, 4 signals clearly differentiate M. massiliense from the other 2 subspecies, M. abscessus and M. bolletii. Moreover, 6 of the 42 M. massiliense isolates showed a spectral pattern similar to M. abscessus. These isolates correspond to the distinctive class of M. massiliense (cluster D) which is closer to M. abscessus by the previous variable number tandem repeat analysis. These results indicate that MALDI-TOF MS is not only useful for the identification of 3 subspecies of M. abscessus complex but also capable of distinguishing clusters of M. massiliense.

  10. The crystal structure of an HSL-homolog EstE5 complex with PMSF reveals a unique configuration that inhibits the nucleophile Ser144 in catalytic triads.

    Science.gov (United States)

    Nam, Ki Hyun; Kim, Soo-Jin; Priyadarshi, Amit; Kim, Hyun Sook; Hwang, Kwang Yeon

    2009-11-13

    The esterase/lipase family (EC 3.1.1.3/EC 3.1.1.1) represents a diverse group of hydrolases that catalyze the cleavage of ester bonds and are widely distributed in animals, plants and microorganisms. Among these enzymes, hormone-sensitive lipases, play a critical role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes. Recently, we reported the structural and biological characterization of EstE5 from the metagenome library [K.H. Nam, M.Y. Kim, S.J. Kim, A. Priyadarshi, W.H. Lee, K.Y. Hwang, Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase, Biochem. Biophys. Res. Commun. 379 (2009) 553-556]. The structure of this protein revealed that it belongs to the HSL-family. Here, we report the inhibition of the activity of the HSL-homolog EstE5 protein as determined by the use of esterase/lipase inhibitors. Our results revealed that the EstE5 protein is significantly inhibited by PMSF. In addition, this is the first study to identify the crystal structures of EstE5-PMSF at 2.4 and 2.5A among the HSL-homolog structures. This structural configuration is similar to that adopted when serine proteases are inhibited by PMSF. The results presented here provide valuable information regarding the properties of the HSL-family.

  11. Fine-mapping analysis revealed complex pleiotropic effect and tissue-specific regulatory mechanism of TNFSF15 in primary biliary cholangitis, Crohn's disease and leprosy.

    Science.gov (United States)

    Sun, Yonghu; Irwanto, Astrid; Toyo-Oka, Licht; Hong, Myunghee; Liu, Hong; Andiappan, Anand Kumar; Choi, Hyunchul; Hitomi, Yuki; Yu, Gongqi; Yu, Yongxiang; Bao, Fangfang; Wang, Chuan; Fu, Xian; Yue, Zhenhua; Wang, Honglei; Zhang, Huimin; Kawashima, Minae; Kojima, Kaname; Nagasaki, Masao; Nakamura, Minoru; Yang, Suk-Kyun; Ye, Byong Duk; Denise, Yosua; Rotzschke, Olaf; Song, Kyuyoung; Tokunaga, Katsushi; Zhang, Furen; Liu, Jianjun

    2016-08-10

    Genetic polymorphism within the 9q32 locus is linked with increased risk of several diseases, including Crohn's disease (CD), primary biliary cholangitis (PBC) and leprosy. The most likely disease-causing gene within 9q32 is TNFSF15, which encodes the pro-inflammatory cytokine TNF super-family member 15, but it was unknown whether these disparate diseases were associated with the same genetic variance in 9q32, and how variance within this locus might contribute to pathology. Using genetic data from published studies on CD, PBC and leprosy we revealed that bearing a T allele at rs6478108/rs6478109 (r(2) = 1) or rs4979462 was significantly associated with increased risk of CD and decreased risk of leprosy, while the T allele at rs4979462 was associated with significantly increased risk of PBC. In vitro analyses showed that the rs6478109 genotype significantly affected TNFSF15 expression in cells from whole blood of controls, while functional annotation using publicly-available data revealed the broad cell type/tissue-specific regulatory potential of variance at rs6478109 or rs4979462. In summary, we provide evidence that variance within TNFSF15 has the potential to affect cytokine expression across a range of tissues and thereby contribute to protection from infectious diseases such as leprosy, while increasing the risk of immune-mediated diseases including CD and PBC.

  12. Characterization of the Complex Locus of Bean Encoding Polygalacturonase-Inhibiting Proteins Reveals Subfunctionalization for Defense against Fungi and Insects1

    Science.gov (United States)

    D'Ovidio, Renato; Raiola, Alessandro; Capodicasa, Cristina; Devoto, Alessandra; Pontiggia, Daniela; Roberti, Serena; Galletti, Roberta; Conti, Eric; O'Sullivan, Donal; De Lorenzo, Giulia

    2004-01-01

    Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects. PMID:15299124

  13. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation.

    Science.gov (United States)

    Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T; Reger, Albert S; Sankaran, Banumathi; Casteel, Darren E; Herberg, Friedrich W; Kim, Choel

    2016-05-03

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.

  14. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  15. Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

    Science.gov (United States)

    Cordina, Nicole M; Liew, Chu K; Potluri, Phani R; Curmi, Paul M; Fajer, Piotr G; Logan, Timothy M; Mackay, Joel P; Brown, Louise J

    2014-01-01

    The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

  16. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Science.gov (United States)

    Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

    2014-01-01

    Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any

  17. Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

    Directory of Open Access Journals (Sweden)

    Nicole M Cordina

    Full Text Available The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+ sensor Troponin complex (Tn, is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI subunit upon the release of Ca2+ from Troponin C (cTnC. Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57, the inhibitory region (cTnI143, and two sites on the regulatory switch region (cTnI151 and cTnI159. Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å away from the hydrophobic N-lobe of troponin C (cTnC